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 ( 89 Zr: t 1/2 = 78.4 h, β + : 22.8%, E β+max = 901 keV; EC: 77%, E γ = 909 keV) to improve disease detection, identify patients for individualized therapeutic interventions. and monitor their response to those interventions. However, release of the 89 Zr 4+ 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, 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 [ 89 Zr]Zr-PCTA and [ 89 Zr]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.

JSL is the Director of the MSK Radiochemistry and Molecular Imaging Probe Core that provides contract, development and manufacturing services for ImaginAb. N.P. is/has served as a consultant/advisory board and has received honoraria for Actinium Pharma, Progenics, Medimmune/ Astrazeneca, Illumina, Imaginab, and conducts research supported by Ymabs, Imaginab, BMS, Bayer, Clarity pharma, Janssen and Regeneron.

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis. Methods: Acid-sensitive cyclic peptide c[E 4 W 5 C] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with 64 Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [ 64 Cu]Cu-c[E 4 W 5 C] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non-pH-sensitive cell-penetrating control peptide (c[R 4 W 5 C]) was used to confirm specificity of [ 64 Cu]Cu-c[E 4 W 5 C]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [ 64 Cu]Cu-c[E 4 W 5 C] PET signal. Results: [ 64 Cu]Cu-c[E 4 W 5 C] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/ nmol. Significantly increased [ 64 Cu]Cu-c[E 4 W 5 C] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines (P , 0.001-0.05). Additionally, there was significantly increased [ 64 Cu]Cu-c[E 4 W 5 C] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [ 64 Cu] Cu-c[R 4 W 5 C] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [ 64 Cu]Cu-c[E 4 W 5 C] tracer. Conclusion: Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [ 64 Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

Epidermal-specific deletion of the homeobox transcription regulator DLX3 disrupts keratinocyte differentiation and results in an IL-17-linked psoriasis-like skin inflammation. To identify the epidermal initiating signals produced by DLX3-null keratinocytes, we performed acute deletion of DLX3 in adult epidermis using a tamoxifen-inducible Krt14-cre/ERT system. K14CreERT;DLX3 skin exhibited dysregulated expression of differentiation-associated genes, upregulation of proinflammatory cytokines, and accumulation of Langerhans cells and macrophages within 3 days of tamoxifen-induced DLX3 ablation. We also observed increased accumulation of IL-17A-secreting Vγ4 γδ T cells and heightened levels of IL-17 and IL-36 family of cytokines starting 1 week after DLX3 deletion. Interestingly, transcriptome profiling of K14CreERT;DLX3 epidermis at 3 days identified activated STAT3 as a transcriptional regulator and revealed differential expression of STAT3 signaling-related genes. Furthermore, activation of STAT3 was strongly increased in K14CreERT;DLX3 skin, and topical treatment with an inhibitor of STAT3 activation attenuated the immune phenotype. RNA-seq analysis of vehicle and STAT3 inhibitor treated K14CreERT;DLX3 skin identified differentially expressed genes associated with inhibition of leukocyte infiltration. Collectively, our results show that DLX3 is a critical regulator of STAT3 signaling network that maintains skin homeostasis.