Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

Gallium Uncouples Iron Metabolism to Enhance Glioblastoma Radiosensitivity.

Gallium-based therapy has been considered a potentially effective cancer therapy for decades and has recently re-emerged as a novel therapeutic strategy for the management of glioblastoma tumors. Gallium targets the iron-dependent phenotype associated with aggressive tumors by mimicking iron in circulation and gaining intracellular access through transferrin-receptor-mediated endocytosis. Mechanistically, it is believed that gallium inhibits critical iron-dependent enzymes like ribonucleotide reductase and NADH dehydrogenase (electron transport chain complex I) by replacing iron and removing the ability to transfer electrons through the protein secondary structure. However, information regarding the effects of gallium on cellular iron metabolism is limited. As mitochondrial iron metabolism serves as a central hub of the iron metabolic network, the goal of this study was to investigate the effects of gallium on mitochondrial iron metabolism in glioblastoma cells. Here, it has been discovered that gallium nitrate can induce mitochondrial iron depletion, which is associated with the induction of DNA damage. Moreover, the generation of gallium-resistant cell lines reveals a highly unstable phenotype characterized by impaired colony formation associated with a significant decrease in mitochondrial iron content and loss of the mitochondrial iron uptake transporter, mitoferrin-1. Moreover, gallium-resistant cell lines are significantly more sensitive to radiation and have an impaired ability to repair any sublethal damage and to survive potentially lethal radiation damage when left for 24 h following radiation. These results support the hypothesis that gallium can disrupt mitochondrial iron metabolism and serve as a potential radiosensitizer.

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.

Pharmacological ascorbate as a novel therapeutic strategy to enhance cancer immunotherapy.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

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.

Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

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.

Progress in Targeted Alpha-Particle-Emitting Radiopharmaceuticals as Treatments for Prostate Cancer Patients with Bone Metastases.

Bone metastasis remains a major cause of death in cancer patients, and current therapies for bone metastatic disease are mainly palliative. Bone metastases arise after cancer cells have colonized the bone and co-opted the normal bone remodeling process. In addition to bone-targeted therapies (e.g., bisphosphonate and denosumab), hormone therapy, chemotherapy, external beam radiation therapy, and surgical intervention, attempts have been made to use systemic radiotherapy as a means of delivering cytocidal radiation to every bone metastatic lesion. Initially, several bone-seeking beta-minus-particle-emitting radiopharmaceuticals were incorporated into the treatment for bone metastases, but they failed to extend the overall survival in patients. However, recent clinical trials indicate that radium-223 dichloride (223RaCl2), an alpha-particle-emitting radiopharmaceutical, improves the overall survival of prostate cancer patients with bone metastases. This success has renewed interest in targeted alpha-particle therapy development for visceral and bone metastasis. This review will discuss (i) the biology of bone metastasis, especially focusing on the vicious cycle of bone metastasis, (ii) how bone remodeling has been exploited to administer systemic radiotherapies, and (iii) targeted radiotherapy development and progress in the development of targeted alpha-particle therapy for the treatment of prostate cancer bone metastasis.

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.

Imaging and dosimetric characteristics of 67 Cu.

In recent years the use of beta-emitting radiopharmaceuticals for cancer therapy has expanded rapidly following development of therapeutics for neuroendocrine tumors, prostate cancer, and other oncologic malignancies. One emerging beta-emitting radioisotope of interest for therapy is 67Cu (t1/2: 2.6 d) due to its chemical equivalency with the widely-established positron-emitting isotope 64Cu (t1/2: 12.7 h). In this work we evaluate both the imaging and dosimetric characteristics of 67Cu, as well as producing the first report of SPECT/CT imaging using 67Cu. To this end, 67Cu was produced by photon-induced reactions on isotopically-enriched 68Zn at the Low-Energy Accelerator Facility (LEAF) of Argonne National Laboratory, followed by bulk separation of metallic 68Zn by sublimation and radiochemical purification by column chromatography. Gamma spectrometry was performed by efficiency-calibrated high-purity germanium (HPGe) analysis to verify absolute activity calibration and establish radionuclidic purity. Absolute activity measurements corroborated manufacturer-recommended dose-calibrator settings and no radionuclidic impurities were observed. Using the Clinical Trials Network anthropomorphic chest phantom, SPECT/CT images were acquired. Medium energy (ME) SPECT collimation was found to provide the best image quality from the primary 185 keV gamma emission of 67Cu. Reconstructed images of 67Cu were similar in quality to images acquired using 177Lu. Recovery coefficients were calculated and compared against quantitative images of 99mTc, 177Lu, and 64Cu within the same anthropomorphic chest phantom. Production and clinical imaging of 67Cu appears feasible, and future studies investigating the therapeutic efficacy of 67Cu-based radiopharmaceuticals are warranted.

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.

Biodistribution and Multicompartment Pharmacokinetic Analysis of a Targeted α Particle Therapy.

Targeted α particle therapy (TAT) is ideal for treating disease while minimizing damage to surrounding nontargeted tissues due to short path length and high linear energy transfer (LET). We developed a TAT for metastatic uveal melanoma, targeting the melanocortin-1 receptor (MC1R), which is expressed in 94% of uveal melanomas. Two versions of the therapy are being investigated: 225Ac-DOTA-Ahx-MC1RL (225Ac-Ahx) and 225Ac-DOTA-di-d-Glu-MC1RL (225Ac-di-d-Glu). The biodistribution (BD) from each was studied and a multicompartment pharmacokinetic (PK) model was developed to describe drug distribution rates. Two groups of 16 severe combined immunodeficient (SCID) mice bearing high MC1R expressing tumors were intravenously injected with 225Ac-Ahx or 225Ac-di-d-Glu. After injection, four groups (n = 4) were euthanized at 24, 96, 144, and 288 h time points for each cohort. Tumors and 13 other organs were harvested at each time point. Isomeric γ spectra were measured in tissue samples using a scintillation γ detector and converted to α activity using factors for γ ray abundance per α decay. Time activity curves were calculated for each organ. A five-compartment PK model was built with the following compartments: blood, tumor, normal tissue, kidney, and liver. This model is characterized by a system of five ordinary differential equations using mass action kinetics, which describe uptake, intercompartmental transitions, and clearance rates. The ordinary differential equations were simultaneously solved and fit to experimental data using a genetic algorithm for optimization. The BD data show that both compounds have minimal distribution to organs at risk other than the kidney and liver. The PK parameter estimates had less than 5% error. From these data, 225Ac-Ahx showed larger and faster uptake in the liver. Both compounds had comparable uptake and clearance rates for other compartments. The BD and PK behavior for two targeted radiopharmaceuticals were investigated. The PK model fit the experimental data and provided insight into the kinetics of the compounds systematically.

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.

A Monte Carlo Method for Determining the Response Relationship between Two Commonly Used Detectors to Indirectly Measure Alpha Particle Radiation Activity.

Using targeted ligands to deliver alpha-emitting radionuclides directly to tumor cells has become a promising therapeutic strategy. To calculate the radiation dose to patients, activities of parent and daughter radionuclides must be measured. Scintillation detectors can be used to quantify these activities; however, activities found in pre-clinical and clinical studies can exceed their optimal performance range. Therefore, a method of correcting scintillation detector measurements at higher activities was developed using Monte Carlo modeling. Because there are currently no National Institute of Standards and Technology traceable Actinium-225 (225Ac) standards available, a well-type ionization chamber was used to measure 70.3 ± 7.0, 144.3 ± 14.4, 222.0 ± 22.2, 299.7 ± 30.0, 370.0 ± 37.0, and 447.7 ± 44.7 kBq samples of 225Ac obtained from Oak Ridge National Lab. Samples were then placed in a well-type NaI(Tl) scintillation detector and spectra were obtained. Alpha particle activity for each species was calculated using gamma abundance per alpha decay. MCNP6 Monte Carlo software was used to simulate the 4π-geometry of the NaI(Tl) detector. Using the ionization chamber reading as activity input to the Monte Carlo model, spectra were obtained and compared to NaI(Tl) spectra. Successive simulations of different activities were run until a spectrum minimizing the mean percent difference between the two was identified. This was repeated for each sample activity. Ionization chamber calibration measurements showed increase in error from 3% to 10% as activities decreased, resulting from decreasing detection efficiency. Measurements of 225Ac using both detector types agreed within 7% of Oak Ridge stated activities. Simulated Monte Carlo spectra of 225Ac were successfully generated. Activities obtained from these spectra differed with ionization chamber readings up to 156% at 147.7 kBq. Simulated spectra were then adjusted to correct NaI(Tl) measurements to be within 1%. These were compared to ionization chamber readings and a response relationship was determined between the two instruments. Measurements of 225Ac and daughter activity were conducted using a NaI(Tl) scintillation detector calibrated for energy and efficiency and an ionization chamber calibrated for efficiency using a surrogate calibration reference. Corrections provided by Monte Carlo modeling improve the accuracy of activity quantification for alpha-particle emitting radiopharmaceuticals in pre-clinical and clinical studies.

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.

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.