Metallaphotoredox aryl and alkyl radiomethylation for PET ligand discovery.

Positron emission tomography (PET) radioligands (radioactively labelled tracer compounds) are extremely useful for in vivo characterization of central nervous system drug candidates, neurodegenerative diseases and numerous oncology targets1. Both tritium and carbon-11 radioisotopologues are generally necessary for in vitro and in vivo characterization of radioligands2, yet there exist few radiolabelling protocols for the synthesis of either, inhibiting the development of PET radioligands. The synthesis of such radioligands also needs to be very rapid owing to the short half-life of carbon-11. Here we report a versatile and rapid metallaphotoredox-catalysed method for late-stage installation of both tritium and carbon-11 into the desired compounds via methylation of pharmaceutical precursors bearing aryl and alkyl bromides. Methyl groups are among the most prevalent structural elements found in bioactive molecules, and so this synthetic approach simplifies the discovery of radioligands. To demonstrate the breadth of applicability of this technique, we perform rapid synthesis of 20 tritiated and 10 carbon-11-labelled complex pharmaceuticals and PET radioligands, including a one-step radiosynthesis of the clinically used compounds [11C]UCB-J and [11C]PHNO. We further outline the direct utility of this protocol for preclinical PET imaging and its translation to automated radiosynthesis for routine radiotracer production in human clinical imaging. We also demonstrate this protocol for the installation of other diverse and pharmaceutically useful isotopes, including carbon-14, carbon-13 and deuterium.

Exploring Aqueous Coordination Chemistry of Highly Lewis Acidic Metals with Emerging Isotopes for Nuclear Medicine.

Nuclear medicine harnesses radioisotopes for the diagnosis and treatment of disease. While the isotopes 99mTc and 111In have enabled the clinical diagnosis of millions of patients over the past 3 decades, more recent clinical translation of numerous 68Ga/177Lu-based radiopharmaceuticals for diagnostic imaging and therapy underscores the clinical utility of metal-based radiopharmaceuticals in mainstream cancer treatment. In addition to such established radionuclides, advancements in radioisotope production have enabled the production of radionuclides with a broad range of half-lives and emission properties of interest for nuclear medicine. Chemical means to form kinetically inert, in vivo-compatible species that can be modified with disease-targeting vectors is imperative. This presents a challenge for radiosiotopes of elements where the aqueous chemistry is still underdeveloped and poorly understood. Here, we discuss our efforts to date in exploring the aqueous, radioactive coordination chemistry of highly Lewis acidic metal ions and how our discoveries apply to the diagnosis and treatment of cancer in preclinical models of disease. The scope of this Account includes approaches to aqueous coordination of to-date understudied highly Lewis acidic metal ions with radioisotopes of emerging interest and the modulation of well-understood coordination environments of radio-coordination complexes to induce metal-catalyzed reactivity for separation and pro-drug applications.First, we discuss the development of seven-coordinate, small-cavity macrocyclic chelator platform mpatcn/picaga as an exemplary case study, which forms robust complexes with 44Sc/47Sc isotopes. Due to the high chemical hardness and pronounced Lewis acidity of the Sc3+ ion, the displacement of ternary ligand H2O by 18/natF- can be achieved to form an inert Sc-18/natF bond. Corresponding coordination complex natSc-18F is in vivo compatible and forms a theranostic tetrad with corresponding 44Sc/47Sc, 177Lu complexes all exhibiting homologous biodistribution profiles. Another exceptionally hard, highly Lewis acidic ion with underdeveloped aqueous chemistry and emerging interest in nuclear medicine is 45Ti4+. To develop de novo approaches to the mononuclear chelation of this ion under aqueous conditions, we employed a fragment-based bidentate ligand screening approach which identified two leads. The screen successfully predicted the formation of [45Ti][Ti(TREN-CAM)], a Ti-triscatechol complex that exhibits remarkable in vivo stability. Furthermore, the fragment-based screen also identified approaches that enabled solid-phase separation of Ti4+ and Sc3+ of interest in streamlining the isotope production of 45Ti and accessing new ways to separate 44Ti/44Sc for the development of a long-lived generator system. In addition to establishing the inert chelation of Ti4+ and Sc3+, we introduce controlled, metal-induced reactivity of corresponding coordination complexes on macroscopic and radiotracer scales. Metal-mediated autolytic amide bond cleavage (MMAAC) enables the temperature-dependent release of high-molar-activity, ready-to-inject radiopharmaceuticals; cleavage is selectively triggered by coordinated trivalent Lewis acid nat/68Ga3+ or Sc3+. Following the scope of reactivity and mechanistic studies, we validated MMAAC for the synthesis of high-molar-activity radiopharmaceuticals to image molecular targets with low expression and metal-mediated prodrug hydrolysis in vivo.This Account summarizes how developing the aqueous coordination chemistry and tuning the chemical reactivity of metal ions with high Lewis acidity at the macroscopic and tracer scales directly apply to the radiopharmaceutical synthesis with clinical potential.

Diagnostic Positron Emission Tomography Imaging with Zirconium-89 Desferrioxamine B Squaramide: From Bench to Bedside.

Molecular imaging with antibodies radiolabeled with positron-emitting radionuclides combines the affinity and selectivity of antibodies with the sensitivity of Positron Emission Tomography (PET). PET imaging allows the visualization and quantification of the biodistribution of the injected radiolabeled antibody, which can be used to characterize specific biological interactions in individual patients. This characterization can provide information about the engagement of the antibody with a molecular target such as receptors present in elevated levels in tumors as well as providing insight into the distribution and clearance of the antibody. Potential applications of clinical PET with radiolabeled antibodies include identifying patients for targeted therapies, characterization of heterogeneous disease, and monitoring treatment response.Antibodies often take several days to clear from the blood pool and localize in tumors, so PET imaging with radiolabeled antibodies requires the use of a radionuclide with a similar radioactive half-life. Zirconium-89 is a positron-emitting radionuclide that has a radioactive half-life of 78 h and relatively low positron emission energy that is well suited to radiolabeling antibodies. It is essential that the zirconium-89 radionuclide be attached to the antibody through chemistry that provides an agent that is stable in vivo with respect to the dissociation of the radionuclide without compromising the biological activity of the antibody.This Account focuses on our research using a simple derivative of the bacterial siderophore desferrioxamine (DFO) with a squaramide ester functional group, DFO-squaramide (DFOSq), to link the chelator to antibodies. In our work, we produce conjugates with an average ∼4 chelators per antibody, and this does not compromise the binding of the antibody to the target. The resulting antibody conjugates of DFOSq are stable and can be easily radiolabeled with zirconium-89 in high radiochemical yields and purity. Automated methods for the radiolabeling of DFOSq-antibody conjugates have been developed to support multicenter clinical trials. Evaluation of several DFOSq conjugates with antibodies and low molecular weight targeting agents in tumor mouse models gave PET images with high tumor uptake and low background. The promising preclinical results supported the translation of this chemistry to human clinical trials using two different radiolabeled antibodies. The potential clinical impact of these ongoing clinical trials is discussed.The use of DFOSq to radiolabel relatively low molecular weight targeting molecules, peptides, and peptide mimetics is also presented. Low molecular weight molecules typically clear the blood pool and accumulate in target tissue more rapidly than antibodies, so they are usually radiolabeled with positron-emitting radionuclides with shorter radioactive half-lives such as fluorine-18 (t1/2 ∼ 110 min) or gallium-68 (t1/2 ∼ 68 min). Radiolabeling peptides and peptide mimetics with zirconium-89, with its longer radioactive half-life (t1/2 = 78 h), could facilitate the centralized manufacture and distribution of radiolabeled tracers. In addition, the ability to image patients at later time points with zirconium-89 based agents (e.g. 4-24 h after injection) may also allow the delineation of small or low-uptake disease sites as the delayed imaging results in increased clearance of the tracer from nontarget tissue and lower background signal.

From cyclotrons to chromatography and beyond: a guide to the production and purification of theranostic radiometals.

Recent clinical success with metal-based radiopharmaceuticals has sparked an interest in the potential of these drugs for personalized medicine. Although often overlooked, the success and global impact of nuclear medicine is contingent upon the purity and availability of medical isotopes, commonly referred to as radiometals. For nuclear medicine to reach its true potential and change patient lives, novel production and purification techniques that increase inventory of radiometals are desperately needed. This tutorial review serves as a resource for those both new and experienced in nuclear medicine by providing a detailed explanation of the foundations for the production and purification of radiometals, stemming from nuclear physics, analytical chemistry, and so many other fields, all in one document. The fundamental science behind targetry, particle accelerators, nuclear reactors, nuclear reactions, and radiochemical separation are presented in the context of the field. Finally, a summary of the latest breakthroughs and a critical discussion of the threats and future potential of the most utilized radiometals is also included. With greater understanding of the fundamentals, fellow scientists will be able to better interpret the literature, identify knowledge gaps or problems and ultimately invent new production and purification pathways to increase the global availability of medical isotopes.

Moving Beyond Isothiocyanates: A Look at the Stability of Conjugation Links Toward Radiolysis in 89Zr-Labeled Immunoconjugates.

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 89Zr-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 89Zr-labeled conjugates, as well as on the aggregation of the conjugates.

Surgical radioguidance with beta-emitting radionuclides; challenges and possibilities: A position paper by the EANM.

Radioguidance that makes use of ß-emitting radionuclides is gaining in popularity and could have potential to strengthen the range of existing radioguidance techniques. While there is a strong tendency to develop new PET radiotracers, due to favorable imaging characteristics and the success of theranostics research, there are practical challenges that need to be overcome when considering use of ß-emitters for surgical radioguidance. In this position paper, the EANM identifies the possibilities and challenges that relate to the successful implementation of ß-emitters in surgical guidance, covering aspects related to instrumentation, radiation protection, and modes of implementation.

Brachytherapy at the nanoscale with protein functionalized and intrinsically radiolabeled [169Yb]Yb2O3 nanoseeds.

PURPOSE: Classical brachytherapy of solid malignant tumors is an invasive procedure which often results in an uneven dose distribution, while requiring surgical removal of sealed radioactive seed sources after a certain period of time. To circumvent these issues, we report the synthesis of intrinsically radiolabeled and gum Arabic glycoprotein functionalized [169Yb]Yb2O3 nanoseeds as a novel nanoscale brachytherapy agent, which could directly be administered via intratumoral injection for tumor therapy. METHODS: 169Yb (T½ = 32 days) was produced by neutron irradiation of enriched (15.2% in 168Yb) Yb2O3 target in a nuclear reactor, radiochemically converted to [169Yb]YbCl3 and used for nanoparticle (NP) synthesis. Intrinsically radiolabeled NP were synthesized by controlled hydrolysis of Yb3+ ions in gum Arabic glycoprotein medium. In vivo SPECT/CT imaging, autoradiography, and biodistribution studies were performed after intratumoral injection of radiolabeled NP in B16F10 tumor bearing C57BL/6 mice. Systematic tumor regression studies and histopathological analyses were performed to demonstrate therapeutic efficacy in the same mice model. RESULTS: The nanoformulation was a clear solution having high colloidal and radiochemical stability. Uniform distribution and retention of the radiolabeled nanoformulation in the tumor mass were observed via SPECT/CT imaging and autoradiography studies. In a tumor regression study, tumor growth was significantly arrested with different doses of radiolabeled NP compared to the control and the best treatment effect was observed with ~ 27.8 MBq dose. In histopathological analysis, loss of mitotic cells was apparent in tumor tissue of treated groups, whereas no significant damage in kidney, lungs, and liver tissue morphology was observed. CONCLUSIONS: These results hold promise for nanoscale brachytherapy to become a clinically practical treatment modality for unresectable solid cancers.

89Zr-immunoPET-guided selection of a CD33xIL15 fusion protein optimized for antitumor immune cell activation and in vivo tumour retention in acute myeloid leukaemia.

PURPOSE: Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). METHODS: We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. RESULTS: Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). CONCLUSION: This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.

Targeting of immune checkpoint regulator V-domain Ig suppressor of T-cell activation (VISTA) with 89Zr-labelled CI-8993.

BACKGROUND: CI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial. METHODS: CI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours. RESULTS: Stable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [89Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice. CONCLUSIONS: We radiolabelled and validated [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.

Radiolabelling and preclinical characterisation of [89Zr]Zr-Df-ATG-101 bispecific to PD-L1/4-1BB.

PURPOSE: ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo. METHODS: ATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [89Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [89Zr]Zr-labelled antibodies. The specificity of [89Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies. RESULTS: The Df-conjugation and [89Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [89Zr]Zr-Df-ATG-101 and [89Zr]Zr-Df-anti-PD-L1. Tumour uptake of [89Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo. CONCLUSION: [89Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [89Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [89Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

Evaluation of a novel 177Lu-labelled therapeutic Affibody molecule with a deimmunized ABD domain and improved biodistribution profile.

PURPOSE: Fusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required. METHODS: The HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with 177Lu. Affinity, specificity, and in vivo targeting properties of [177Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated. RESULTS: The maximum molar activity of 52 GBq/µmol [177Lu]Lu-PEP49989 was obtained. [177Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [177Lu]Lu-PEP49989 was similar to the affinity of [177Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [177Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [177Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [177Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [177Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild. CONCLUSION: In preclinical studies, [177Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

Development of a Specifically Labeled 89Zr Antibody for the Noninvasive Imaging of Tumors Overexpressing B7-H3.

B7-H3 has emerged as a promising target and potential biomarker for diagnosing tumors, evaluating treatment efficacy, and determining patient prognosis. Hu4G4 is a recombinant humanized antibody that selectively targets the extracellular domain of human B7-H3. In this study, we describe the radiolabeling of hu4G4 with the positron emission tomography (PET) emitter radionuclide zirconium 89 (89Zr) and evaluate its potency as an immuno-PET tracer for B7-H3-targeted imaging by comparing it in vitro and in vivo to [89Zr]Zr-DFO-DS-5573a using various models. The radiolabeled compound, [89Zr]Zr-desferrioxamine-hu4G4 ([89Zr]Zr-DFO-hu4G4), demonstrated a high radiochemical purity (RCP) of greater than 99% and a specific activity of 74 MBq/mg following purification. Additionally, it maintained stability in human serum albumin (HSA) and acetate buffer, preserving over 90% of its RCP after 7 days. Three cell lines targeting human B7-H3(U87/CT26-CD276/GL261-CD276) were used. Flow cytometry analysis indicated that the B7-H3-positive cells (U87/CT26-CD276/GL261-CD276) had a higher B7-H3 protein level with no expression in the B7-H3-negative cells (CT26-wt/GL261-wt) (P < 0.001). Moreover, the cellular uptake was 45.71 ± 3.78% for [89Zr]Zr-DFO-hu4G4 in CT26-CD276 cells versus only 0.93 ± 0.47% in CT26-wt cells and 30.26 ± 0.70% when [89Zr]Zr-DFO-hu4G4 in CT26-CD276 cells were blocked with 100× 8H9. The cellular uptake of [89Zr]Zr-DFO-hu4G4 was akin to that observed with [89Zr]Zr-DFO-DS-5573a with no significant differences (45.71 ± 3.78 % vs 47.07 ± 0.86 %) in CT26-CD276 cells. Similarly, the CT26-CD276 mouse model demonstrated markedly low organ uptake and elevated tumor uptake 48 h after [89Zr]Zr-DFO-hu4G4 injection. PET/CT analysis showed that the tumor-to-muscle (T/M) ratios were substantially higher compared to other imaging groups: 27.65 ± 3.17 in CT26-CD276 mice versus 11.68 ± 4.19 in CT26-wt mice (P < 0.001) and 16.40 ± 0.78 when 100× 8H9 was used to block [89Zr]Zr-DFO-hu4G4 in CT26-CD276 mice (P < 0.01) at 48 h post-injection. Additionally, the tracer showed markedly high accumulation in the tumor region (22.57 ± 3.03% ID/g), comparable to the uptake of [89Zr]Zr-DFO-DS-5573a (24.76 ± 5.36% ID/g). A dosimetry estimation study revealed that the effective dose for [89Zr]Zr-DFO-hu4G4 was 2.96 × 10-01 mSv/MBq, which falls within the acceptable range for further research in nuclear medicine. Collectively, these results indicated that [89Zr]Zr-DFO-hu4G4 was successfully fabricated and applied in B7-H3-targeted tumor PET/CT imaging, which showed excellent imaging quality and tumor detection efficacy in tumor-bearing mice. It is a promising imaging agent for identifying tumors that overexpress B7-H3 for future clinical applications.

Construction of [89Zr]Zr-Labeled HuL13 for ImmunoPET Imaging of LAG-3 Checkpoint Expression on Tumor-Infiltrating T Cells.

Lymphocyte activation gene 3 (LAG-3) has attracted much attention as a potentially valuable immune checkpoint. Individual identification of LAG-3 expression at screening and during treatment could improve the successful implementation of anti-LAG-3 therapies. HuL13 is a human IgG1 monoclonal antibody that binds to the LAG-3 receptor in T cells. Here, we used [89Zr]Zr-labeled HuL13 to delineate LAG-3+ T-cell infiltration into tumors via positron emission tomography (PET) imaging. A549/LAG-3 cells, which stably express LAG-3, were generated by infection with lentivirus. The uptake of [89Zr]Zr-DFO-HuL13 in A549/LAG-3 cells was greater than that in the negative control (A549/NC) cells at each time point. The equilibrium dissociation constant (Kd) of [89Zr]Zr-DFO-HuL13 for the LAG-3 receptor was 8.22 nM. PET imaging revealed significant uptake in the tumor areas of A549/LAG-3 tumor-bearing mice from 24 h after injection (SUVmax = 2.43 ± 0.06 at 24 h). As a proof of concept, PET imaging of the [89Zr]Zr-DFO-HuL13 tracer was further investigated in an MC38 tumor-bearing humanized LAG-3 mouse model. PET imaging revealed that the [89Zr]Zr-DFO-HuL13 tracer specifically targets human LAG-3 expressed on tumor-infiltrating lymphocytes (TILs). In addition to the tumors, the spleen was also noticeably visible. Tumor uptake of the [89Zr]Zr-DFO-HuL13 tracer was lower than its uptake in the spleen, but high uptake in the spleen could be reduced by coinjection of unlabeled antibodies. Coinjection of unlabeled antibodies increases tracer activity in the blood pool, thereby improving tumor uptake. Dosimetry evaluation of the healthy mouse models revealed that the highest absorbed radiation dose was in the spleen, followed by the liver and heart wall. In summary, these studies demonstrate the feasibility of using the [89Zr]Zr-DFO-HuL13 tracer for the detection of LAG-3 expression on TILs. Further clinical evaluation of the [89Zr]Zr-DFO-HuL13 tracer may be of significant help in the stratification and management of patients suitable for anti-LAG-3 therapy.

Ultrastable PLGA-Coated 177Lu-Microspheres for Radioembolization Therapy of Hepatocellular Carcinoma.

Transarterial radioembolization (TARE) is a highly effective localized radionuclide therapy that has been successfully used to treat hepatocellular carcinoma (HCC). Extensive research has been conducted on the use of radioactive microspheres (MSs) in TARE, and the development of ideal radioactive MSs is crucial for clinical trials and patient treatment. This study presents the development of a radioactive MS for TARE of HCC. These MSs, referred to as 177Lu-MS@PLGA, consist of poly(lactic-co-glycolic acid) (PLGA) copolymer and radioactive silica MSs, labeled with 177Lu and then coated with PLGA. It has an extremely high level of radiostability. Cellular experiments have shown that it can cause DNA double-strand breaks, leading to cell death. In vivo radiostability of 177Lu-MS@PLGA is demonstrated by microSPECT/CT imaging. In addition, the antitumor study has shown that TARE of 177Lu-MS@PLGA can effectively restrain tumor growth without harmful side effects. Thus, 177Lu-MS@PLGA exhibits significant potential as a radioactive MS for the treatment of HCC.

PET Imaging of Differentiated Thyroid Cancer with TSHR-Targeted [89Zr]Zr-TR1402.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, ß+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Development and Evaluation of DOTA-FAPI-Maleimide as a Novel Radiotracer for Tumor Theranostic with Extended Circulation.

This study aimed to evaluate a novel albumin-binding strategy for addressing the challenge of insufficient tumor retention of fibroblast activation protein inhibitors (FAPIs). Maleimide, a molecule capable of covalent binding to free thiol groups, was modified to conjugate with FAPI-04 in order to enhance its binding to endogenous albumin, resulting in an extended blood circulation half-life and increased tumor uptake. DOTA-FAPI-maleimide was prepared and radiolabeled with Ga-68 and Lu-177, followed by cellular assays, pharmacokinetic analysis, PET/CT, and SPECT/CT imaging to assess the probe distribution in various tumor-bearing models. Radiolabeling of the modified probe was successfully achieved with a radiochemical yield of over 99% and remained stable for 144 h. Cellular assays showed that the ligand concentration required for 50% inhibition of the probe was 1.20 ± 0.31 nM, and the Kd was 0.70 ± 0.07 nM with a Bmax of 7.94 ± 0.16 fmol/cell, indicative of higher specificity and affinity of DOTA-FAPI-maleimide compared to other FAPI-04 variants. In addition, DOTA-FAPI-maleimide exhibited a persistent blood clearance half-life of 7.11 ± 0.34 h. PET/CT images showed a tumor uptake of 2.20 ± 0.44%ID/g at 0.5 h p.i., with a tumor/muscle ratio of 5.64 in HT-1080-FAP tumor-bearing models. SPECT/CT images demonstrated long-lasting tumor retention. At 24 h p.i., the tumor uptake of [177Lu]Lu-DOTA-FAPI-maleimide reached 5.04 ± 1.67%ID/g, with stable tumor retention of 3.40 ± 1.95%ID/g after 4 days p.i. In conclusion, we developed and evaluated the thiol group-attaching strategy, which significantly extended the circulation and tumor retention of the adapted FAPI tracer. We envision its potential application for clinical cancer theranostics.

Novel [68Ga/177Lu]Ga/Lu-AZ-093 as PSMA-Targeting Agent for Diagnosis and Radiotherapy.

Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer cells can serve as a target for imaging and radioligand therapy (RLT). Previously, [68Ga]Ga-P16-093, containing a Ga(III) chelator, N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), displayed excellent PSMA-targeting properties and showed a high tumor uptake and retention useful for diagnosis in prostate cancer patients. Recently, [177Lu]Lu-PSMA-617 has been approved by the U.S. food and drug administration (FDA) for the treatment of prostate cancer patients. Derivatives of PSMA-093 using AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), as the chelator, were designed as alternative agents forming complexes with both diagnostic and therapeutic radiometals, such as gallium-68 (log K = 22.18) or lutetium-177 (log K = 21.85). The aim of this study is to evaluate AAZTA-Gly-O-(methylcarboxy)-Tyr-Phe-Lys-NH-CO-NH-Glu (designated as AZ-093, 1) leading to a gallium-68/lutetium-177 theranostic pair as potential PSMA targeting agents. Synthesis of the desired precursor, AZ-093, 1, was effectively accomplished. Labeling with either [68Ga]GaCl3 or [177Lu]LuCl3 in a sodium acetate buffer solution (pH 4-5) at 50 °C in 5 to 15 min produced either [68Ga]Ga-1 or [177Lu]Lu-1 with high yields and excellent radiochemical purities. Results of in vitro binding studies, cell uptake, and retention (using PSMA-positive prostate carcinoma cells line, 22Rv1-FOLH1-oe) were comparable to that of [68Ga]Ga-P16-093 and [177Lu]Lu-PSMA-617, respectively. Specific cellular uptake was determined with or without the competitive blocking agent (2 µM of "cold" PSMA-11). Cellular binding and internalization showed a time-dependent increase over 2 h at 37 °C in the PSMA-positive cells. The cell uptakes were completely blocked by the "cold" PSMA-11 suggesting that they are competing for the same PSMA binding sites. In the mouse model with implanted PSMA-positive tumor cells, both [68Ga]Ga-1 and [177Lu]Lu-1 displayed excellent uptake and retention in the tumor. Results indicate that [68Ga]Ga/[177Lu]Lu-1 (68Ga]Ga/[177Lu]Lu-AZ-093) is potentially useful as PSMA-targeting agent for both diagnosis and radiotherapy of prostate cancer.

Investigating the In Vivo Biodistribution of Extracellular Vesicles Isolated from Various Human Cell Sources Using Positron Emission Tomography.

Positron emission tomography (PET) is a powerful tool for investigating the in vivo behavior of drug delivery systems. We aimed to assess the biodistribution of extracellular vesicles (EVs), nanosized vesicles secreted by cells isolated from various human cell sources using PET. EVs were isolated from mesenchymal stromal cells (MSCs) (MSC EVs), human macrophages (Mϕ EVs), and a melanoma cell line (A375 EVs) by centrifugation and were conjugated with deferoxamine for radiolabeling with Zr-89. PET using conjugated and radiolabeled EVs evaluated their in vivo biodistribution and tissue tropisms. Our study also investigated differences in mouse models, utilizing immunocompetent and immunocompromised mice and an A375 xenograft tumor model. Lastly, we investigated the impact of different labeling techniques on the observed EV biodistribution, including covalent surface modification and membrane incorporation. PET showed that all tested EVs exhibited extended in vivo circulation and generally low uptake in the liver, spleen, and lungs. However, Mϕ EVs showed high liver uptake, potentially attributable to the intrinsic tissue tropism of these EVs from the surface protein composition. MSC EV biodistribution differed between immunocompetent and immunodeficient mice, with increased spleen uptake observed in the latter. PET using A375 xenografts demonstrated efficient tumor uptake of EVs, but no preferential tissue-specific tropism of A375 EVs was found. Biodistribution differences between labeling techniques showed that surface-conjugated EVs had preferential blood circulation and low liver, spleen, and lung uptake compared to membrane integration. This study demonstrates the potential of EVs as effective drug carriers for various diseases, highlights the importance of selecting appropriate cell sources for EV-based drug delivery, and suggests that EV tropism can be harnessed to optimize therapeutic efficacy. Our findings indicate that the cellular source of EVs, labeling technique, and animal model can influence the observed biodistribution.

Effect of Ibuprofen as an Albumin Binder on Melanoma-Targeting Properties of 177Lu-Labeled Ibuprofen-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

The purpose of this study was to examine how the introduction of ibuprofen (IBU) affected tumor-targeting and biodistribution properties of 177Lu-labeled IBU-conjugated alpha-melanocyte-stimulating hormone peptides. The IBU was used as an albumin binder and conjugated to the DOTA-Lys moiety without or with a linker to yield DOTA-Lys(IBU)-GG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(IBU)-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex peptides. Their melanocortin-receptor 1 (MC1R) binding affinities were determined on B16/F10 melanoma cells first. Then the biodistribution of 177Lu-labeled peptides was determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to choose the lead peptide for further examination. The full biodistribution and melanoma imaging properties of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex were further evaluated using B16/F10 melanoma-bearing C57 mice. DOTA-Lys(IBU)-GG-Nle-CycMSHhex, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex displayed the IC50 values of 1.41 ± 0.37, 1.52 ± 0.08, 0.03 ± 0.01, and 0.58 ± 0.06 nM on B16/F10 melanoma cells, respectively. 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex exhibited the lowest liver and kidney uptake among all four designed 177Lu peptides. Therefore, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was further evaluated for its full biodistribution and melanoma imaging properties. The B16/F10 melanoma uptake of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was 19.5 ± 3.12, 24.12 ± 3.35, 23.85 ± 2.08, and 10.80 ± 2.89% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Moreover, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex could clearly visualize the B16/F10 melanoma lesions at 2 h postinjection. The conjugation of IBU with or without a linker to GGNle-CycMSHhex affected the MC1R binding affinities of the designed peptides. The charge of the linker played a key role in the liver and kidney uptake of 177Lu-Asp-IBU, 177Lu-Asn-IBU, and 177Lu-Dab-IBU. 177Lu-Asp-IBU exhibited higher tumor/liver and tumor/kidney uptake ratios than those of 177Lu-Asn-IBU and 177Lu-Dab-IBU, underscoring its potential evaluation for melanoma therapy in the future.

Capturing Mercury-197m/g for Auger Electron Therapy and Cancer Theranostic with Sulfur-Containing Cyclen-Based Macrocycles.

The interest in mercury radioisotopes, 197mHg (t1/2 = 23.8 h) and 197gHg (t1/2 = 64.14 h), has recently been reignited by the dual diagnostic and therapeutic nature of their nuclear decays. These isotopes emit γ-rays suitable for single photon emission computed tomography imaging and Auger electrons which can be exploited for treating small and metastatic tumors. However, the clinical utilization of 197m/gHg radionuclides is obstructed by the lack of chelators capable of securely binding them to tumor-seeking vectors. This work aims to address this challenge by investigating a series of chemically tailored macrocyclic platforms with sulfur-containing side arms, namely, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), and 1,7-bis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,10-diacetic acid (DO2A2S). 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetracetic acid (DOTA), the widest explored chelator in nuclear medicine, and the nonfunctionalized backbone 1,4,7,10-tetrazacyclododecane (cyclen) were considered as well to shed light on the role of the sulfanyl arms in the metal coordination. To this purpose, a comprehensive experimental and theoretical study encompassing aqueous coordination chemistry investigations through potentiometry, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and density functional theory (DFT) calculations, as well as concentration- and temperature-dependent [197m/gHg]Hg2+ radiolabeling and in vitro stability assays in human serum was conducted. The obtained results reveal that the investigated chelators rapidly complex Hg2+ in aqueous media, forming extremely thermodynamically stable 1:1 metal-to-ligand complexes with superior stabilities compared to those of DOTA or cyclen. These complexes exhibited 6- to 8-fold coordination environments, with donors statically bound to the metal center, as evidenced by the presence of 1H-199Hg spin-spin coupling via NMR. A similar octacoordinated environment was also found for DOTA in both solution and solid state, but in this case, multiple slowly exchanging conformers were detected at ambient temperature. The sulfur-rich ligands quantitatively incorporate cyclotron-produced [197m/gHg]Hg2+ under relatively mild reaction conditions (pH = 7 and T = 50 °C), with the resulting radioactive complexes exhibiting decent stability in human serum (up to 75% after 24 h). By developing viable chelators and understanding the impact of structural modifications, our research addresses the scarcity of suitable chelating agents for 197m/gHg, offering promise for its future in vivo application as a theranostic Auger-emitter radiometal.