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Surface modification of materials with proteins has various biological applications, and hence the methodology for surface modification needs to accommodate a wide range of proteins that differ in structure, size, and function. Presented here is a methodology that uses the Affinity Bioorthogonal Chemistry (ABC) tag, 3-(2-pyridyl)-6-methyltetrazine (PyTz), for the site-selective modification and purification of proteins and subsequent attachment of the protein to trans -cyclooctene (TCO)-functionalized hydrogel microfibers. This method of surface modification is shown to maintain the functionality of the protein after conjugation with proteins of varying size and functionalities, namely, HaloTag, NanoLuc luciferase (NanoLuc), and fibronectin type III domains 9-10 (FNIII 9-10). The method also supports surface modification with multiple proteins, which is shown by the simultaneous conjugation of HaloTag and NanoLuc on the microfiber surface. The ability to control the relative concentrations of multiple proteins presented on the surface is shown with the use of HaloTag and superfolder GFP (sfGFP). This application of the ABC-tagging methodology expands on existing surface modification methods and provides flexibility in the site-selective protein conjugation methods used along with the rapid kinetics of tetrazine ligation.

There have been predictions that the use of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in zirconium-89 ( 89 Zr) immuno-positron emission tomography ( 89 Zr-immunoPET) could enhance the in vivo stability of 89 Zr radioimmunoconjugates. However, conjugating [ 89 Zr]Zr-DOTA to a monoclonal antibody (mAb) remains a challenge as the heat treatment required for [ 89 Zr]Zr-DOTA chelation can lead to thermal denaturation of the mAb moieties. We developed a method for synthesizing [ 89 Zr]Zr-DOTA-mAb based on a tetrazine (Tz)-conjugated bifunctional DOTA derivative 2,2',2″-(10-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,21,26-trioxo-6,9,12,15,18-pentaoxa-29-carboxy-2,22,25-triazanonacosane-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-Tz) and the inverse electron-demand Diels-Alder (IEDDA) click chemistry reaction where trans -cyclooctene-modified mAbs are conjugated to [ 89 Zr]Zr-DOTAGA without being exposed to heat. The stability of IEDDA-derived [ 89 Zr]Zr-DOTAGA-trastuzumab was confirmed by in vitro, ex vivo, and in vivo testing and comparative analysis against the conventional deferoxamine (DFO) counterpart [ 89 Zr]Zr-DFO-trastuzumab. The in vivo immunoPET imaging using [ 89 Zr]Zr-DOTAGA-trastuzumab clearly visualized human epidermal growth factor receptor 2-positive malignancies in murine xenograft models. Greater tumor contrast was observed from [ 89 Zr]Zr-DOTAGA-trastuzumab at a 72-h delayed scan compared with [ 89 Zr]Zr-DFO-trastuzumab. These findings suggest that our IEDDA ligation approach can be an effective means of synthesizing [ 89 Zr]Zr-DOTA-mAb and can enhance the theranostic potential of 89 Zr-immunoPET in DOTA-mediated radioimmunotherapy.

Objective : About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG 2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64 Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2- S -(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64 Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [ 64 Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [ 18 F]FLT PET. Immuno-PET with [ 64 Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [ 64 Cu]CuCl 2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [ 64 Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein ( n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein ( n = 3). PET imaging revealed high uptake of [ 64 Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV) mean reached 4.70 ± 0.42 and 5.37 ± 0.40 ( n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 ( n = 4; p < 0.001). In the metastatic model, the following SUV mean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 ( n = 4) to 3.67 ± 0.33% ID/g ( n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 ( n = 4) to 8.35 ± 1.30% ID/g ( n = 5; p < 0.001), respectively. Conclusions: [ 64 Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.

Bioorthogonal chemistry has become a mainstay in chemical biology and is making inroads in the clinic with recent advances in protein targeting and drug release. Since the field's beginning, a major focus has been on designing bioorthogonal reagents with good selectivity, reactivity, and stability in complex biological environments. More recently, chemists have imbued reagents with new functionalities like click-and-release or light/enzyme-controllable reactivity. We have previously developed a controllable cyclopropene-based bioorthogonal ligation, which has excellent stability in physiological conditions and can be triggered to react with tetrazines by exposure to enzymes, biologically significant small molecules, or light spanning the visual spectrum. Here, to improve reactivity and gain a better understanding of this system, we screened diene reaction partners for the cyclopropene. We found that a cyclopropene-quinone pair is 26 times faster than reactions with 1,2,4,5-tetrazines. Additionally, we showed that the reaction of the cyclopropene-quinone pair can be activated by two orthogonal mechanisms: caging group removal on the cyclopropene and oxidation/reduction of the quinone. Finally, we demonstrated that this caged cyclopropene-quinone can be used as an imaging tool to label the membranes of fixed, cultured cells., (© 2024 Wiley-VCH GmbH.)

Light-activated fluorescence represents a potent tool for investigating subcellular structures and dynamics, offering enhanced control over the temporal and spatial aspects of the fluorescence signal. While alkyl-substituted tetrazine has previously been reported as a photo-trigger for various fluorophore scaffolds, its limited photochemical efficiency and high activation energy have constrained its widespread application at the biomolecular level. In this study, we demonstrate that a single sulfur atom substitution of tetrazine greatly enhances the photochemical properties of tetrazine conjugates and significantly improves their photocleavage efficiency. Notably, the resulting sulfur-tetrazine can be activated using a lower-energy light source, thus transforming it into a valuable visible-light photo-trigger. To introduce this photo-trigger into biological systems, we have developed a series of visible-light activatable small molecular dyes, along with a photoactivatable noncanonical amino acid containing sulfur-tetrazine. Using the Genetic Code Expansion technology, this novel amino acid is genetically incorporated into fluorescent protein molecules, serving as a phototrigger to create an innovative photoactivatable protein. These advancements in tetrazine-scaffold photo-trigger design open up new avenues for generating photoactivatable biomolecules, promising to greatly facilitate the exploration of biological functions and structures.

Fibroblast activation protein (FAP) is specifically expressed on cancer-associated fibroblasts in over 90% of tumors and is considered a promising target for cancer theranostics. Here, we developed a novel tracer, DOTA-FAPT, and labeled it with gallium-68 and lutetium-177 as a theranostic pair. [ 68 Ga]Ga/[ 177 Lu]Lu-FAPT exhibited high stability and hydrophilicity, as well as strong affinity to the FAP target. Micro-PET/CT imaging revealed that [ 68 Ga]Ga-FAPT exhibited significantly increased uptake in tumors and extended retention in A549-FAP and U87MG tumor xenografts as compared to [ 68 Ga]Ga-FAPI-04, demonstrating favorable pharmacokinetic characteristics in vivo. Therapeutic studies showed that [ 177 Lu]Lu-FAPT had higher tumor accumulation compared to [ 177 Lu]Lu-FAPI-04, leading to stronger tumor growth inhibition. The first-in-human evaluation also revealed that [ 68 Ga]Ga-FAPT has good in vivo distribution and superior diagnostic efficacy on primary and lymph node metastases in a patient with lung cancer. Our encouraging results suggest that 68 Ga/ 177 Lu-labeled DOTA-FAPT is a theranostic pair with broad application prospect.

Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and β-emitting isotope 177 Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S -2-(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [ 177 Lu]Lu-Gemini was prepared with no-carrier-added 177 LuCl 3 to a molar-specific activity of 123 GBq/μmol and radiochemical purity of more than 99%. The specificity of BsAb- 177 Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [ 177 Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [ 177 Lu]Lu- S -2-(4-aminobenzyl)-DOTA ([ 177 Lu]Lu-DOTA-Bn) in naïve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/anti-DOTA IgG-scFv BsAb, a clearing agent, and [ 177 Lu]Lu-Gemini) in mouse models. Results: Initial in vivo studies showed that [ 177 Lu]Lu-Gemini behaved similarly to [ 177 Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [ 177 Lu]Lu-Gemini to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [ 177 Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor-to-normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [ 177 Lu]Lu-Gemini in DOTA-PRIT leads to improved TIs and augmented [ 177 Lu]Lu-Gemini tumor uptake and retention in comparison to monovalent [ 177 Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor-bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [ 177 Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [ 177 Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [ 177 Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [ 177 Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered 177 Lu activity while still achieving high TIs for both the blood (>100) and the kidneys (>30)., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: 18 F-labelled somatostatin receptor (SSTR) analogs offer several advantages over 68 Ga in terms of yield, cost, spatial resolution and detection rate. This study presents an interim analysis of a prospective trial designed to assess the safety, biodistribution and dosimetry of [ 18 F]AlF-NOTA-LM3, and compare its diagnostic efficacy and clinical management outcomes with [ 68 Ga]Ga-DOTATATE or [ 68 Ga]Ga-NODAGA-LM3 in patients with well-differentiated NETs., Methods: Twenty-one patients with histologically confirmed well-differentiated neuroendocrine tumors (G1 and G2) were prospectively recruited. The first eight patients underwent serial PET scans at 5, 15, 30, 45, 60, and 120 min after [ 18 F]AlF-NOTA-LM3 injection to assess biodistribution and dosimetry. The remaining patients underwent whole-body PET/CT scans. [ 18 F]AlF-NOTA-LM3 and [68Ga]Ga-DOTATATE PET/CT were done within a week, with a minimum 24-hour interval between the two scans. Focal uptake above the surrounding background activity and could not be explained by physiologic uptake was considered lesions of NETs. Lesion number, tumor uptake, and tumor-to-background ratio (TBR) were compared. In patients with discrepant findings, the size of the smallest lesions (measured on coregistered CT) detected on [ 68 Ga]Ga-DOTATATE and [ 18 F]AlF-NOTA-LM3 was compared., Results: [ 18 F]AlF-NOTA-LM3 was safe and well-tolerated. Physiological uptake of [ 18 F]AlF-NOTA-LM3 was significantly lower than that of [ 68 Ga]Ga-DOTATATE in abdominal organs and bone marrow, but higher in blood pool and lung. The mean effective dose was 0.024 ± 0.014 mSv/MBq. [ 18 F]AlF-NOTA-LM3 detected significantly more liver lesions (457 vs. 291, P = 0.006) and lymph node lesions (30 vs. 22, P = 0.011) compared to [ 68 Ga]Ga-DOTATATE. The tumor uptake was comparable, but TBR was significantly higher with [ 18 F]AlF-NOTA-LM3 for lesions from all sites except for the duodenum. The size of the minimum liver lesions (0.54 ± 0.15 vs. 1.01 ± 0.49, P<0.001) and lymph node lesions (0.50 ± 0.19 vs. 1.26 ± 0.86, P = 0.024) detected on [ 18 F]ALF-NOTA-LM3 were significantly smaller than those detected on [ 68 Ga]Ga-DOTATATE., Conclusion: [ 18 F]AlF-NOTA-LM3 shows favorable biodistribution, higher spatial resolution and superior performance than [ 68 Ga]Ga-DOTATATE in detecting liver and lymph node metastases, with higher TBR. Notably, it is the first SSTR analog to show superiority in detecting lymph node lesions when compared to [ 68 Ga]Ga-DOTATATE., Trial Registration: ClinicalTrials.gov identifier NCT06056362., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Triggering receptor expressed on myeloid cells 2 (TREM2) plays an essential role in microglia activation and is being investigated as a potential therapeutic target for modulation of microglia in several neurological diseases. In this study, we present the development and preclinical evaluation of 64 Cu-labeled antibody-based PET radiotracers as tools for non-invasive assessment of TREM2 expression. Furthermore, we tested the potential of an antibody transport vehicle (ATV) that binds human transferrin receptor to facilitate transcytosis of TREM2 antibody-based radiotracers to the CNS and improve target engagement. Methods: A TREM2 antibody with an engineered transport vehicle (ATV:4D9) and without (4D9) were covalently modified with p NCS-benzyl-NODAGA and labeled with copper-64. Potency, stability, and specificity were assessed in vitro followed by in vivo PET imaging at the early 2 h, intermediate 20 h, and late imaging time points 40 h post-injection using a human transferrin receptor (hTfR) expressing model for amyloidogenesis (5xFAD;TfR mu/hu ) or wild-type mice (WT;TfR mu/hu ), and hTfR negative controls. Organs of interest were isolated to determine biodistribution by ex vivo autoradiography. Cell sorting after in vivo tracer injection was used to demonstrate cellular specificity for microglia and to validate TREM2 PET results in an independent mouse model for amyloidogenesis (App SAA ;TfR mu/hu ). For translation to human imaging, a human TREM2 antibody (14D3) was radiolabeled and used for in vitro autoradiography on human brain sections. Results: The 64 Cu-labeled antibodies were obtained in high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity. Antibody modification did not impact TREM2 binding. ATV:4D9 binding proved to be specific, and the tracer stability was maintained over 48 h. The uptake of [ 64 Cu]Cu-NODAGA-ATV:4D9 in the brains of hTfR expressing mice was up to 4.6-fold higher than [ 64 Cu]Cu-NODAGA-4D9 in mice without hTfR. TREM2 PET revealed elevated uptake in the cortex of 5xFAD mice compared to wild-type, which was validated by autoradiography. PET-to-biodistribution correlation revealed that elevated radiotracer uptake in brains of 5xFAD;TfR mu/hu mice was driven by microglia-rich cortical and hippocampal brain regions. Radiolabeled ATV:4D9 was selectively enriched in microglia and cellular uptake explained PET signal enhancement in App SAA ;TfR mu/hu mice. Human autoradiography showed elevated TREM2 tracer binding in the cortex of patients with Alzheimer's disease. Conclusion: [ 64 Cu]Cu-NODAGA-ATV:4D9 has potential for non-invasive assessment of TREM2 as a surrogate marker for microglia activation in vivo . ATV engineering for hTfR binding and transcytosis overcomes the blood-brain barrier restriction for antibody-based PET radiotracers. TREM2 PET might be a versatile tool for many applications beyond Alzheimer's disease, such as glioma and chronic inflammatory diseases., Competing Interests: Competing Interests: G.C.P., D.X., and K.M.M. are full-time employees and shareholders of Denali Therapeutics. The other authors have declared that no competing interest exists., (© The author(s).)

Multistate nonadiabatic dynamics combined with Mixed-Reference Spin-Flip Time-Dependent Density Functional Theory (MRSF-TDDFT) were performed to investigate the chemoexcitation dynamics of firefly dioxetanone (FDO - in S 0 ) to oxyluciferin (OxyLH - in S 1 ) and its subsequent decay dynamics. The formation of oxyluciferin occurs within approximately 100 fs and is primarily controlled by oscillatory CO 2 decarboxylation. Unexpected radiationless decay from oxyluciferin was also observed, facilitated by intramolecular rotation. Simulations under three thermal conditions reveal that higher initial thermal energy not only enhances the formation of oxyluciferin but also increases radiationless decay by surpassing barriers to the ground state. Conversely, lower thermal energy conditions reduce oxyluciferin formation but suppress radiationless decay. These findings suggest that optimal conditions for higher chemiluminescence quantum yield involve initial high thermal energy to accelerate CO 2 decarboxylation and gradual thermal dissipation to prevent intramolecular rotation of oxyluciferin. This approach could enhance the chemiluminescence quantum yield beyond the current limit of 40%, offering significant potential for applications in biological imaging and analytical chemistry.

B-cell maturation antigen (BCMA) has emerged as a promising tumor marker for the diagnosis and treatment of multiple myeloma. The noninvasive and rapid detection of BCMA expression in vivo provides significant value in screening and evaluating multiple myeloma patients receiving BCMA-targeted therapy. We identified the BCMA-targeting peptide BP1 from a one-bead-one-compound (OBOC) peptide library using a high-throughput microarray strategy. The BCMA-targeting specificity and affinity of BP1 were assessed by surface plasmon resonance imaging (SPRi), flow cytometry, and confocal imaging. BCMA-positive (H929) and BCMA-negative (K562) subcutaneous tumor models were established and labeled with 68 Ga for BP1, followed by PET imaging and biodistribution studies. PET imaging demonstrated that 68 Ga-labeled BP1 has significant specific uptake in multiple myeloma, enabling rapid identification of BCMA expression and precise delineation of the disease. Thus, BP1 represents an ideal candidate for multiple myeloma imaging.

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 K d was 0.70 ± 0.07 nM with a B max 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 [ 177 Lu]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.

Introduction: Zirconium-89 ( 89 Zr) is a positron emitter with several advantages over other shorter-lived positron emission tomography (PET) compatible radiometals such as gallium-68 or copper-64. These include practically unlimited availability, extremely low cost, greatly facilitated distribution logistics, positron energy fit for medical PET imaging, and sufficiently long physical half-life to enable PET imaging at later time points for patient-specific dosimetry estimations. Despite these apparent benefits, the reception of 89 Zr in the nuclear medicine community has been tepid. The driving factor for the absence of broader adaptation is mostly routed in its final formulation - [ 89 Zr]zirconium oxalate. While serving as a suitable precursor solution for the gold standard chelator deferoxamine (DFO), [ 89 Zr]Zr-oxalate is inaccessible for the most commonly used chelators, such as the macrocyclic DOTA, due to its pre-chelated state. Consequently, pioneering work has been conducted by multiple research groups to create oxalate-free forms of [ 89 Zr]Zr 4+ , either via chemical conversion of oxalate into other counterion forms or via direct radiochemical isolation of [ 89 Zr]ZrCl 4 , showing that [ 89 Zr]Zr-DOTA complexes are possible and stable. However, this success was accompanied by challenges, including complex and labor-intensive radiochemical processing and radiolabeling procedures as well as the relatively minuscule conversion rates. Here, we report on the direct production of [ 89 Zr]ZrCl 4 avoiding oxalate and metal contaminants to enable efficient radiolabeling of DOTA constructs., Methods: We based our direct production of [ 89 Zr]ZrCl 4 on previously reported methods and further optimized its quality by including an additional iron-removing step using the TK400 Resin. Here, we avoided using oxalic acid and effectively minimized the content of trace metal contaminants. Our two-step purification procedure was automated, and we confirmed excellent radionuclide purity, minimal trace metals content, great reactivity over time, and high specific molar activity. In addition, DOTA-based PSMA-617 and DOTAGA-based PSMA-I&T were radiolabeled to demonstrate the feasibility of direct radiolabeling and to estimate the maximum apparent specific activities. Lastly, the biodistribution of [ 89 Zr]Zr-PSMA-617 was assessed in mice bearing PC3-PIP xenografts, and the results were compared to the previously published data., Results: A total of 18 batches, ranging from 6.9 to 20 GBq (186 to 541 mCi), were produced. The specific molar activity for [ 89 Zr]ZrCl 4 exceeded 0.96 GBq (26 mCi) per nanomole of zirconium. The radionuclidic purity was >99 %, and the trace metals content was in the <1 ppm range. The [ 89 Zr]ZrCl 4 remained in its reactive chemical form for at least five days when stored in cyclic olefin polymer (COP) vials. Batches of 11.1 GBq (300 mCi) of [ 89 Zr]Zr-PSMA-617 and 14.4 GBq (390 mCi) of [ 89 Zr]Zr-PSMA-I&T, corresponding to specific activities of 11.1 MBq/μg (0.3 mCi/μg), and 14.4 MBq/μg (0.39 mCi/μg), respectively, were produced. [ 89 Zr]Zr-PSMA-617 animal PET imaging results were in agreement with the previously published data., Conclusion: In this work, we report on a suitable application of TK400 Resin to remove iron during [ 89 Zr]ZrCl 4 radiochemical isolation. The breakthrough allows for direct radiolabeling of DOTA-based constructs with [ 89 Zr]ZrCl 4 , leading to high apparent molar activities and excellent conversion rates., Competing Interests: Declaration of competing interest Serge K. Lyashchenko declares that he serves as a consultant to Ymabs Therapeutics, Inc.; serves as a consultant to Solve Tx, Inc.; and serves as senior advisor to, is co-founder of, and has stock options in Evergreen Theragnostics, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: The unsatisfactory efficacy of PD-L1 antibodies in glioblastoma (GBM) is largely due to the temporal and spatial heterogeneity of PD-L1 expression. Molecular imaging can enhance understanding of the tumor immune microenvironment and guide immunotherapy. However, highly sensitive imaging agents capable of effectively visualizing PD-L1 heterogeneity are limited. This study introduces a novel PET tracer, offering improved imaging of PD-L1 heterogeneity in GBM xenografts, with a comparative analysis to [ 18 F]AlF-NOTA-WL12., Methods: [ 18 F]AlF-NOTA-PCP2 was synthesized with high purity and its affinity for PD-L1 was characterized using surface plasmon resonance (SPR) and cell binding assays. Its specificity for PD-L1 was evaluated both in vitro using various cell lines and in vivo with GBM xenograft models in NOD/SCID mice. PET/CT imaging was conducted to evaluate the tracer's biodistribution, pharmacokinetics, and ability to quantify tumoral spatial heterogeneity of PD-L1 expression. A focused comparative analysis between [ 18 F]AlF-NOTA-PCP2 and [ 18 F]AlF-NOTA-WL12 was conducted, examining binding affinity, biodistribution, pharmacokinetics, and imaging effectiveness in GBM xenografts. Additionally, human radiation dosimetry estimates compared the safety profiles of both tracers., Results: [ 18 F]AlF-NOTA-PCP2 demonstrated high radiochemical purity (> 95%) and a strong affinity for PD-L1, comparable to [ 18 F]AlF-NOTA-WL12. In vitro and in vivo studies confirmed its specificity for PD-L1, with increased uptake in PD-L1 expressing cells and tumors. Toxicological profiles indicated no significant abnormalities in serum biochemical indicators or major organ tissues. MicroPET/CT imaging showed [ 18 F]AlF-NOTA-PCP2's effectiveness in visualizing PD-L1 expression levels and spatial heterogeneity in GBM xenografts. Comparative studies revealed [ 18 F]AlF-NOTA-PCP2's improved pharmacokinetic properties, including higher tumor-to-blood ratios and lower nonspecific liver uptake, as well as reduced radiation exposure compared to [ 18 F]AlF-NOTA-WL12., Conclusion: [ 18 F]AlF-NOTA-PCP2 distinguishes itself as an exceptionally sensitive PET/CT tracer, adept at non-invasively and accurately quantifying PD-L1 expression and its spatial heterogeneity in tumors, especially in GBM. Its favorable pharmacokinetic properties, safety profile, and high affinity for PD-L1 highlight its potential for enhancing the precision of cancer immunotherapy and guiding individualized treatment strategies. While promising, its clinical translation, especially in brain imaging, necessitates further validation in clinical trials., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Prostate cancer affects 1 in 6 men, and it is the second‑leading cause of cancer-related death in American men. Surgery is one of the main treatment modalities for prostate cancer, but it often results in incomplete resection margins or complete resection that leads to nerve damage and undesirable side effects. In the present work, we have developed a new bimodal tracer, NODAGA-sCy7.5 PSMAi (prostate-specific membrane antigen inhibitor), labeled with the true matched theranostic pair 64 Cu/ 67 Cu and a near-infrared fluorescent dye. This agent could potentially be used for concomitant PET imaging, optical surgical navigation, and targeted radiopharmaceutical therapy., Methods: A prostate-specific membrane antigen (PSMA)-targeting urea derivative was conjugated to NODAGA for copper radiolabeling and to the near-infrared fluorophore sulfo-Cy7.5 (sCy7.5). Binding studies were performed in PSMA-positive PC-3 PIP cells, as well as uptake and internalization assays in PC-3 PIP cells and PSMA-negative PC-3 wild type cells. Biodistribution studies of the 64 Cu-labeled compound were performed in PC-3 PIP- and PC-3 tumor-bearing mice, and 67 Cu biodistributions of the agent were obtained in PC-3 PIP tumor-carrying mice. PET imaging and fluorescence imaging were also performed, using the same molar doses, in the two mouse models., Results: The PSMA conjugate bound with high affinity to PSMA-positive prostate cancer cells, as opposed to cells that were PSMA-negative. Uptake and internalization were rapid and PSMA-mediated in PC-3 PIP cells, while only minimal non-specific uptake was observed in PC-3 cells. Biodistribution studies showed specific uptake in PC-3 PIP tumors, while accumulation in PC-3 tumor-bearing mice was low. Furthermore, tumor uptake of the 67 Cu-labeled agent in the PC-3 PIP model was statistically equivalent to that of 64 Cu. PET and fluorescence imaging at 0.5 nmol per mouse also demonstrated that PC-3 PIP tumors could be clearly detected, while PC-3 tumors showed no tumor accumulation., Conclusions: NODAGA-sCy7.5-PSMAi was specific and selective in detecting PSMA-positive, as opposed to PSMA-negative, tumors in mouse models of prostate cancer. This bioconjugate could potentially be used for PET staging with 64 Cu, targeted radiopharmaceutical therapy with 67 Cu, and/or image-guided surgery with sCy7.5., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Direct detection of the notorious explosive triacetone triperoxide (TATP) is very difficult because it lacks facile ionization and UV absorbance or fluorescence. Besides, the current indirect methods are time-consuming and need a pre-step for TATP cleavage to hydrogen peroxide. Moreover, they commonly show significant false-positive results in the presence of some camouflage which limits their field applications. Herein, for the first time, a novel label-free field-applicable spectrofluorimetric nanobiosensor was developed for direct TATP detection using a novel activated-protein protected gold nanocluster (ABSA-AuNCs; QY = 28.3 %) synthesized by a combined protein-assisted-ultrasonication procedure., Results: The ABSA-AuNCs revealed a fluorescence spectrum centered at 330.0 nm which was significantly quenched by TATP (binding constant = 154.06 M -1 ; ΔG = -12.5 kJ mol -1 ; E(%) = 88.5 %). This phenomenon was used as a basis for direct TATP quantification, providing a working range of 0.01-40.0 mg L -1 and a detection limit of 6.7 μg L -1 which is the lowest LOD provided for TATP detection up to now. A %RSD of 0.9 % and 1.56 % was obtained for repeatability and inter-day reproducibility, respectively. The selectivity was checked against a variety of camouflages, revealing ultra-selectivity. Several synthetic samples prepared by several camouflages and real samples (clay soil and real water media) were analyzed, revealing quantitative recoveries of TATP., Significance: During the production of the notorious explosive TATP, it can be discharged into water and soil. This novel method eliminated the false-positive results of traditional methods and is applicable for direct quantitative detection of camouflaged TATP and its residues in real soil and water samples in a highly short response time (2 min). The camouflaged TATP analysis is important for tracking the terrorist attacks in field conditions and analysis of soil and water can provide a first indication of the location of the production site., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [ 68 Ga]Ga-NODASA-H8 and [ 68 Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [ 68 Ga]Ga-NODASA-H8 and [ 68 Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (A m , 3.9 ± 0.8 and 34.0 ± 5.3 GBq/μmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [ 68 Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [ 68 Ga]Ga-NODASA-H8 degraded within 30 min. For both 68 Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [ 68 Ga]Ga-NODASA-PH1 is a promising candidate for further in vitro/in vivo evaluation as a tuberculosis-specific infection imaging agent., (© 2024 The Author(s). Journal of Labelled Compounds and Radiopharmaceuticals published by John Wiley & Sons Ltd.)

Bioorthogonal reactions have revolutionized chemical biology by enabling selective chemical transformations within living organisms and cells. This review comprehensively explores bioorthogonal chemistry, emphasizing inverse-electron-demand Diels-Alder (IEDDA) reactions between tetrazines and strained dienophiles and their crucial role in chemical biology and various applications within the human body. This highly reactive and selective reaction finds diverse applications, including cleaving antibody-drug conjugates, prodrugs, proteins, peptide antigens, and enzyme substrates. The versatility extends to hydrogel chemistry, which is crucial for biomedical applications, yet it faces challenges in achieving precise cellularization. In situ activation of cytotoxic compounds from injectable biopolymer belongs to the click-activated protodrugs against cancer (CAPAC) platform, an innovative approach to tumor-targeted prodrug delivery and activation. The CAPAC platform, relying on click chemistry between trans-cyclooctene (TCO) and tetrazine-modified biopolymers, exhibits modularity across diverse tumor characteristics, presenting a promising approach in anticancer therapeutics. The review highlights the importance of bioorthogonal reactions in developing radiopharmaceuticals for positron emission tomography (PET) imaging and theranostics, offering a promising avenue for diverse therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Cholecystokinin 2 receptor (CCK 2 R) is a promising target for imaging and treatment of medullary thyroid cancer due to its overexpression in over 90% of tumor cells. 68 Ga-DOTA-CCK-66 is a recently introduced PET tracer selective for CCK 2 R, which has shown favorable pharmacokinetics in vivo in preclinical experiments. In order to further investigate safety and suitability of this tracer in the human setting, whole-body distribution and radiation dosimetry were evaluated., Patients and Methods: Six patients with a history of medullary thyroid cancer were injected intravenously with 169 ± 19 MBq of 68 Ga-DOTA-CCK-66. Whole-body PET/CT scans were acquired at 10 minutes, 1 hour, 2 hours, and 4 hours after tracer injection. Time-activity curves per organ were determined, and mean organ-absorbed doses and effective doses were calculated using OLINDA/EXM., Results: Injection of a standard activity of 150 MBq of 68 Ga-DOTA-CCK-66 results in an effective dose of 4.5 ± 0.9 mSv. The highest absorbed organ doses were observed in the urinary bladder wall (40 mGy) and the stomach (15 mGy), followed by the kidneys (6 mGy), as well as the liver and the spleen (3 mGy each). CCK 2 R-expressing tumor manifestations could be detected in 2 of the 6 patients, including lymph node, bone, and liver metastases., Conclusions: 68 Ga-DOTA-CCK-66 exhibits a favorable dosimetry. Beyond physiologic receptor expression of the stomach, no other relevant tracer accumulation could be observed, rendering this organ at risk in case of subsequent radioligand therapy using 177 Lu-DOTA-CCK-66., Competing Interests: Conflicts of interest and sources of funding: A patent application on CCK 2 R-targeted compounds including DOTA-CCK-66 with T.G., N.H., C.L., and H.J.W. as inventors has been filed. H.J.W. is founder and shareholder of Scintomics GmbH, Munich, Germany. No other potential conflicts of interest relevant to this article exist. This study has been funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—461577150)., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Radiolabelled puromycin analogues will allow the quantification of protein synthesis through nuclear medicine-based imaging. A particularly useful application could be the non-invasive longitudinal visualisation of mycobacterial activity through direct quantification of puromycin binding. This study assesses the value of [ 68 Ga]Ga-DOTA-puromycin in the visualisation of mycobacteria through positron emission tomography combined with magnetic resonance imaging (µPET/MRI). The radiopharmaceutical was produced by previously published and validated methods. [ 68 Ga]Ga-DOTA-Puromycin imaging was performed on severe immunodeficient mice infected with Bacille Calmette-Guérin-derived M. Bovis (BCG). Acute and chronic infection stages were examined by µPET/MRI. A follow-up group of animals acted as controls (animals bearing S. aureus-derived infection and sterile inflammation) to assess tracer selectivity. [ 68 Ga]Ga-DOTA-puromycin-µPET/MRI images revealed the acute, widespread infection within the right upper shoulder and armpit. Also, [ 68 Ga]Ga-DOTA-puromycin signal sensitivity measured after a 12-week period was lower than that of [ 18 F]FDG-PET in the same animals. A suitable correlation between normalised uptake values (NUV) and gold standard histopathological analysis confirms accurate tracer accumulation in viable bacteria. The radiopharmaceutical showed infection selectivity over inflammation but accumulated in both M. Bovis and S. Aureus, lacking pathogen specificity. Overall, [ 68 Ga]Ga-DOTA-puromycin exhibits potential as a tool for non-invasive protein synthesis visualization, albeit without pathogen selectivity., (© 2024. The Author(s).)

The overexpression of HER2 is pivotal in the initiation and progression of breast cancer. Developing HER2-targeted radiotracers is crucial for noninvasive assessment of HER2 expression, patient selection for HER2-targeted therapy, monitoring treatment response, and identifying resistance. Here, we reported a nonsite-specific coupled radiotracer, 68 Ga-NOTA-SNA004-His 6 , and a site-specific coupled radiotracer, 68 Ga-NODAGA-SNA004-GSC, based on a novel HER2 nanobody, SNA004. Both radiotracers exhibited high affinity, specific targeting, and rapid clearance in vitro and in vivo. Additionally, these tracers and trastuzumab showed noncompetitive binding to HER2. Compared to 68 Ga-NOTA-SNA004-His 6 , 68 Ga-NODAGA-SNA004-GSC demonstrated significantly reduced renal and liver uptake. PET/CT imaging with 68 Ga-NODAGA-SNA004-GSC sensitively detected the responsiveness of various tumor models to trastuzumab and its antibody-drug conjugates (ADCs). Overall, the site-specific coupled radiotracer 68 Ga-NODAGA-SNA004-GSC offered significant advantages in biodistribution and signal-to-noise ratio, making it a valuable tool for monitoring HER2 expression levels before, during, and after trastuzumab and ADC treatment.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG 3 -IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [ 111 In]In or [ 177 Lu]Lu-FAP8-PEG 3 -IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG 3 -IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor-to-healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%-93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [ 177 Lu]Lu-FAP8-PEG 3 -IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Tetrazine-derived fluorogenic labels are extensively studied for their potential in biological and medical imaging. Nonetheless, the fluorescence quenching mechanism in numerous precursors continues to be debated, particularly as the wavelengths extend into the red and near-infrared (NIR) regions. This challenge poses obstacles to systematically optimizing their fluorogenicity, i.e., achieving red-shifted wavelengths and improved fluorescence turn-on signals through click reactions. This paper highlights the significance of photoinduced charge centralization (PCC), a quenching mechanism observed in tetrazine-fused fluorogenic labels with integrated π-conjugations. PCC is primarily responsible for the quenching effects observed in such labels emitting in the red-to-NIR spectrum. Drawing from structure-property relationships, this study proposes two molecular design strategies that incorporate the PCC mechanism and constitutional isomerization to develop high-performance tetrazine-based labels. These strategies facilitate multiplex fluorescence imaging following click reactions, promising significant advancements in bio-orthogonal imaging techniques.

We report the use of DOTA as a chelator for titanium. The resulting complex is fully characterised and in vitro stability studies reveal its high kinetic inertness against transmetallation and transchelation. The radiolabeling of DOTA with 45 Ti, via a guaiacol-based liquid-liquid extraction method, leads to a high radiochemical conversion up to 98%.

Purpose: Pluvicto™ ([ 177 Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2., Methods: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine., Results: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™., Conclusion: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Vascular endothelial growth factor receptor 3 (VEGFR-3) plays a critical role in tumor lymphangiogenesis and metastasis, holding promise as a promising therapeutic target for solid tumors. TMVP1 (LARGR) is a 5-amino acid peptide previously identified in our laboratory from bacterial peptide display system that specifically targets VEGFR-3. Radiolabeled TMVP1 can be used for non-invasive imaging of VEGFR-3 expressing tumors. Homodimeric peptides have better targeting ability than monomeric peptides, and it is worth exploring whether homodimers of TMVP1 ((TMVP1) 2 ) can achieve better imaging effects. This study aimed to explore the peptide properties and tumor assessment value of [ 68 Ga]Ga-labeled (TMVP1) 2 ., Methods: In this study, we developed a TMVP1 homodimer that was conjugated with 1,4,7-triazacyclononane-N, N', N″-triacetic acid (NOTA) via tetraethyleneglycol (PEG 4 ) and triglyicine (Gly 3 ) spacer, and labeled with 68 Ga, to construct [ 68 Ga]Ga-NOTA-(TMVP1) 2 . Binding of VEGFR-3 by TMVP1 and (TMVP1) 2 , respectively, was modeled by molecular docking. The affinity of [ 68 Ga]Ga-NOTA-(TMVP1) 2 for VEGFR-3 and its ability to bind to cells were evaluated. MicroPET imaging and biodistribution studies of [ 68 Ga]Ga-NOTA-(TMVP1) 2 were performed in subcutaneous C33A cervical cancer xenografts. Five healthy volunteers and eight patients with cervical cancer underwent whole-body PET/CT acquisition 30-45 min after intravenous injection of [ 68 Ga]Ga-NOTA-(TMVP1) 2 ., Results: Both molecular docking and cellular experiments showed that homodimeric TMVP1 had a higher affinity for VEGFR-3 than monomeric TMVP1. [ 68 Ga]Ga-NOTA-(TMVP1) 2 was excreted mainly through the renal route and partly through the liver route. In mice bearing C33A xenografts, [ 68 Ga]Ga-NOTA-(TMVP1) 2 specifically localized in the tumor (2.32 ± 0.10% ID/g). Pretreatment of C33A xenograft mice with the unlabeled peptide NOTA-(TMVP1) 2 reduced the enrichment of [ 68 Ga]Ga-NOTA-(TMVP1) 2 in tumors (0.58 ± 0.01% ID/g). [ 68 Ga]Ga-NOTA-(TMVP1) 2 proved to be safe in all healthy volunteers and recruited patients, with no side effects or allergies noted. In cervical cancer patients, a majority of the [ 18 F]-FDG identified lesions (18/22, 81.8%) showed moderate to high signal intensity on [ 68 Ga]Ga-NOTA-(TMVP1) 2. SUVmax and SUVmean were 2.32 ± 0.77 and 1.61 ± 0.48, respectively. With normal muscle (gluteus maximus) as background, tumor-to-background ratios were 3.49 ± 1.32 and 3.95 ± 1.64 based on SUVmax and SUVmean, respectively., Conclusion: The favorable characterizations of [ 68 Ga]Ga-NOTA-(TMVP1) 2 such as convenient synthesis, high specific activity, and high tumor uptake enable the evaluation of VEGFR-3 in cervical cancer patients and warrant further clinical studies., Trial Registration: ChiCTR-DOD-17012458. Registered August 23, 2017 (retrospectively registered)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Despite the inclusion of multiple agents within the prostate cancer treatment landscape, new treatment options are needed to address the unmet need for patients with metastatic castration-resistant prostate cancer (mCRPC). Although prostate-specific membrane antigen is the only cell-surface target to yield clinical benefit in men with advanced prostate cancer, additional targets may further advance targeted immune, cytotoxic, radiopharmaceutical, and other tumor-directed therapies for these patients. Human kallikrein 2 (hK2) is a novel prostate-specific target with little to no expression in nonprostate tissues. This first-in-human phase 0 trial uses an 111 In-radiolabeled anti-hK2 monoclonal antibody, [ 111 In]-DOTA-h11B6, to credential hK2 as a potential target for prostate cancer treatment. Methods: Participants with progressive mCRPC received a single infusion of 2 mg of [ 111 In]-DOTA-h11B6 (185 MBq of 111 In), with or without 8 mg of unlabeled h11B6 to assess antibody mass effects. Sequential imaging and serial blood samples were collected to determine [ 111 In]-DOTA-h11B6 biodistribution, dosimetry, serum radioactivity, and pharmacokinetics. Safety was assessed within a 2-wk follow-up period from the time of [ 111 In]-DOTA-h11B6 administration. Results: Twenty-two participants received [ 111 In]-DOTA-h11B6 and are included in this analysis. Within 6-8 d of administration, [ 111 In]-DOTA-h11B6 visibly accumulated in known mCRPC lesions, with limited uptake in other organs. Two treatment-emergent adverse events unrelated to treatment occurred, including tumor-related bleeding in 1 patient, which led to early study discontinuation. Serum clearance, biodistribution, and tumor targeting were independent of total antibody mass (2 or 10 mg). Conclusion: This first-in-human study demonstrates that tumor-associated hK2 can be identified and targeted using h11B6 as a platform as the h11B6 antibody selectively accumulated in mCRPC metastases with mass-independent clearance kinetics. These data support the feasibility of hK2 as a target for imaging and hK2-directed agents as potential therapies in patients with mCRPC., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: A series of new 68 Ga-labeled tracers based on [ 68 Ga]Ga-PSMA-617 were developed to augment the tumor-to-kidney ratio and reduce the activity accumulation in bladder, ultimately minimize radiation toxicity to the urinary system., Methods: We introduced quinoline group, phenylalanine and decanoic acid into different tracers to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Their binding affinity onto LNCaP cells was determined through in vitro saturation assays and competition binding assays. In vivo metabolic study, PET imaging and biodistribution experiment were performed in LNCaP tumor-bearing B-NSG male mice. The most promising tracer was selected for first-in-human study., Results: Four radiotracers were synthesized with radiochemical purity (RCP) > 95% and molar activity in a range of 20.0-25.5 GBq/μmol. The binding affinities (K i ) of TWS01, TWS02 to PSMA were in the low nanomolar range (< 10 nM), while TWS03 and TWS04 exhibited binding affinities with K i  > 20 nM (59.42 nM for TWS03 and 37.14 nM for TWS04). All radiotracers exhibited high stability in vivo except [ 68 Ga]Ga-TWS03. Micro PET/CT imaging and biodistribution analysis revealed that [ 68 Ga]Ga-TWS02 enabled clear tumor visualization in PET images at 1.5 h post-injection, with higher tumor-to-kidney ratio (T/K, 0.93) and tumor-to-muscle ratio (T/M, 107.62) compared with [ 68 Ga]Ga-PSMA-617 (T/K: 0.39, T/M: 15.01) and [ 68 Ga]Ga-PSMA-11 (T/K: 0.15, T/M: 24.00). In first-in-human study, [ 68 Ga]Ga-TWS02 effectively detected PCa-associated lesions including primary and metastatic lesions, with lower accumulation in urinary system, suggesting that [ 68 Ga]Ga-TWS02 might be applied in the detection of bladder invasion, with minimized radiation toxicity to the urinary system., Conclusion: Introduction of quinoline group, phenylalanine and decanoic acid into different tracers can modulate the binding affinity and pharmacokinetics of PSMA in vivo. [ 68 Ga]Ga-TWS02 showed high binding affinity to PSMA, excellent pharmacokinetic properties and clear imaging of PCa-associated lesions, making it a promising radiotracer for the clinical diagnosis of PCa. Moreover, TWS02 with a chelator DOTA could also label 177 Lu and 225 Ac, which could be used for PCa treatment without significant side effects., Trial Registration: The clinical evaluation of this study was registered On October 30, 2021 at https://www.chictr.org.cn/ (No: ChiCTR2100052545)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fibrinogen-like protein 1 (FGL1) is a potential novel immune checkpoint target for malignant tumor diagnosis and therapy. Accurate detection of FGL1 levels in tumors via noninvasive PET imaging might be beneficial for managing the disease. To achieve this, multiple FGL1-targeting peptides (FGLP) were designed, and a promising candidate, 68 Ga-NOTA-FGLP2, was identified through a high-throughput screening approach using microPET imaging of 68 Ga-labeled peptides. Subsequent in vitro cell experiments showed that uptake values of 68 Ga-NOTA-FGLP2 in FGL1 positive Huh7 tumor cells were significantly higher than those in FGL1 negative U87 MG tumor cells. Further microPET imaging showed that the Huh7 xenografts were clearly visualized with a favorable contrast. ROI analysis showed that the uptake values of the tracer in Huh7 xenografts were 2.63 ± 0.07% ID/g at 30 min p.i.. After treatment with an excess of unlabeled FGLP2, the tumor uptake significantly decreased to 0.54 ± 0.05% ID/g at 30 min p.i.. Moreover, the uptake in U87 MG xenografts was 0.44 ± 0.06% ID/g at the same time point. The tracer was excreted mainly through the renal system. 18 F-FDG PET imaging was also performed in mice bearing Huh7 and U87 MG xenografts, respectively. However, there was no significant difference in the uptake between the tumors with different FGL1 expressions. Preclinical data indicated that 68 Ga-NOTA-FGLP2 might be a suitable radiotracer for in vivo noninvasive visualization of tumors with abundant expression of FGL1. Further investigation of 68 Ga-NOTA-FGLP2 for tumor diagnosis and therapy is undergoing.

High complex stability and longitudinal relaxivity of Gd-based contrast agents are important requirements for magnetic resonance imaging (MRI) because they ensure patient safety and contribute to measurement sensitivity. Charged and zwitterionic Gd 3+ -complexes of the well-known chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) provide an excellent basis for the development of safe and sensitive contrast agents. In this report, we describe the synthesis of DOTA-NOx, a DOTA derivative with four N-oxide functionalities via "click" functionalization of the tetraazide DOTAZA. The resulting complexes Gd-DOTA-NOx and Eu-DOTA-NOx are stable compounds in aqueous solution. NMR-spectroscopic characterization revealed a high excess of the twisted square antiprismatic (TSAP) coordination geometry over square antiprismatic (SAP). The longitudinal relaxivity of Gd-DOTA-NOx was found to be r 1 =7.7 mm -1  s -1 (1.41 T, 37 °C), an unusually high value for DOTA complexes of comparable weight. We attribute this high relaxivity to the steric influence and an ordering effect on outer sphere water molecules surrounding the complex generated by the strongly hydrated N-oxide groups. Moreover, Gd-DOTA-NOx was found to be stable against transchelation with high excess of EDTA (200 eq) over a period of 36 h, and it has a similar in vitro cell toxicity as clinically used DOTA-based GBCAs., (© 2023 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Background: FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi) 2 , labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs., Methods: The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [ 68 Ga]Ga-DOTA.SA.FAPi and [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 . [ 177 Lu]Lu-DOTA.SA.FAPi and [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 , were evaluated in PC3 xenografts. Biodistribution studies of [ 68 Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice., Results: All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 was retained longer in CAFs. [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 and [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 displayed elevated lipophilicity and affinity for human serum proteins compared to [ 68 Ga]Ga-DOTA.SA.FAPi and [ 177 Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 within 3 h compared to [ 68 Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [ 68 Ga]Ga-DOTAGA.(SA.FAPi) 2 versus [ 68 Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 maintained a significant tumor uptake even after 96 h p.i. compared to [ 177 Lu]Lu-DOTA.SA.FAPi., Conclusions: Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC's first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Purpose: Neuropilin-1 (NRP-1) is a multifunctional protein involved in a variety of biological processes such as angiogenesis, tumorigenesis and immunomodulation. It was usually overexpressed in many cancer cell lines and correlated with poor prognosis of breast cancer. Positron emission tomography (PET) is an advanced imaging technique for detecting the function and metabolism of tumor-associated molecules in real time, dynamically, quantitatively and noninvasively. To improve the level of early diagnosis and evaluate the prognosis of breast cancer, an NRP-1 targeting peptide-based tracer [ 68  Ga]Ga-NOTA-PEG 4 -CK2 was designed to sensitively and specifically detect the NRP-1 expression in vivo via PET imaging., Methods: In silico modeling and microscale thermophoresis (MST) assay were carried out to design the NRP-1 targeting peptide NOTA-PEG 4 -CK2, and it was further radiolabeled with 68  Ga to prepare the tracer [ 68  Ga]Ga-NOTA-PEG 4 -CK2. The radiochemical yield (RCY), radiochemical purity (RCP), molar activity (A m ), lipid-water partition coefficient (Log P) and stability of [ 68  Ga]Ga-NOTA-PEG 4 -CK2 were assessed. The targeting specificity of the tracer for NRP-1 was investigated by in vitro cellular uptake assay and in vivo PET imaging as well as blocking studies. The sensitivity of the tracer in monitoring the dynamic changes of NRP-1 expression induced by chemical drug was also investigated in vitro and in vivo. Ex vivo biodistribution, autoradiography, western blot, and immunofluorescence staining were also performed to study the specificity of [ 68  Ga]Ga-NOTA-PEG 4 -CK2 for NRP-1., Results: [ 68  Ga]Ga-NOTA-PEG 4 -CK2 was designed and synthesized with high RCY (> 98%), high stability (RCP > 95%) and high affinity to NRP-1 (K D  = 25.39 ± 1.65 nM). In vitro cellular uptake assay showed that the tracer [ 68  Ga]Ga-NOTA-PEG 4 -CK2 can specifically bind to NRP-1 positive cancer cells MDA-MB-231 (1.04 ± 0.04% at 2 h) rather than NRP-1 negative cancer cells NCI-H1299 (0.43 ± 0.05%). In vivo PET imaging showed the maximum tumor uptake of [ 68  Ga]Ga-NOTA-PEG 4 -CK2 in MDA-MB-231 xenografts (4.16 ± 0.67%ID/mL) was significantly higher than that in NCI-H1299 xenografts (1.03 ± 0.19%ID/mL) at 10 min post injection, and the former exhibited higher tumor-to-muscle uptake ratio (5.22 ± 0.18) than the latter (1.07 ± 0.27) at 60 min post injection. MDA-MB-231 xenografts pretreated with nonradioactive precursor NOTA-PEG 4 -CK2 showed little tumor uptake of [ 68  Ga]Ga-NOTA-PEG 4 -CK2 (1.67 ± 0.38%ID/mL at 10 min post injection). Both cellular uptake assay and PET imaging revealed that NRP-1 expression in breast cancer MDA-MB-231 could be effectively suppressed by SB-203580 treatment and can be sensitively detected by [ 68  Ga]Ga-NOTA-PEG 4 -CK2. Ex vivo analysis also proved the high specificity and sensitivity of [ 68  Ga]Ga-NOTA-PEG 4 -CK2 for NRP-1 expression in MDA-MB-231 xenografts., Conclusion: A promising NRP-1 targeting PET tracer [ 68  Ga]Ga-NOTA-PEG 4 -CK2 was successfully prepared. It showed remarkable specificity and sensitivity in monitoring the dynamic changes of NRP-1 expression. Hence, it could provide valuable information for early diagnosis of NRP-1 relevant cancers and evaluating the prognosis of cancer patients., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Scandium (Sc) isotopes have recently attracted significant attention in the search for new radionuclides with potential uses in personalized medicine, especially in the treatment of specific cancer patient categories. In particular, Sc-43 and Sc-44, as positron emitters with a satisfactory half-life (3.9 and 4.0 h, respectively), are ideal for cancer diagnosis via Positron Emission Tomography (PET). On the other hand, Sc-47, as an emitter of beta particles and low gamma radiation, may be used as a therapeutic radionuclide, which also allows Single-Photon Emission Computed Tomography (SPECT) imaging. As these scandium isotopes follow the same biological pathway and chemical reactivity, they appear to fit perfectly into the "theranostic pair" concept. A step-by-step description, initiating from the moment of scandium isotope production and leading up to their preclinical and clinical trial applications, is presented. Recent developments related to the nuclear reactions selected and employed to produce the radionuclides Sc-43, Sc-44, and Sc-47, the chemical processing of these isotopes and the main target recovery methods are also included. Furthermore, the radiolabeling of the leading chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and its structural analogues with scandium is also discussed and the advantages and disadvantages of scandium complexation are evaluated. Finally, a review of the preclinical studies and clinical trials involving scandium, as well as future challenges for its clinical uses and applications, are presented.

Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [ 177 Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [ 177 Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.

The absence of better biomarkers currently limits early diagnosis and treatment of triple-negative breast cancer (TNBC). Our previously published study reported that the cyclic-peptide SD01 exhibited specific binding to EphA2 (Ephrin type-A receptor 2) on TNBC. To develop a novel PET imaging agent, we prepared gallium-68 ( 68 Ga) labeled-DOTA-SD01 and evaluated its specificity and effectiveness through micro PET/CT imaging in a TNBC-bearing mouse model. SD01 and a control linear peptide YSA were conjugated to DOTA and subsequently labeled with 68 Ga, obtaining 68 Ga-DOTA-SD01 and 68 Ga-DOTA-YSA. Both showed high radiochemical purity, stability, good hydrophilicity, and high binding affinity to 4T1 cells. Micro PET/CT imaging showed high radioactivity accumulation in tumors; SUV mean (mean standardized uptake value) of tumors in the group of 68 Ga-DOTA-SD01 was 3.34 ± 0.25 and 2.65 ± 0.32 in the group of 68 Ga-DOTA-YSA; T/NT ratios (target to non-target, SUV mean ratios of tumor to muscle) were 3.12 ± 0.06 and 2.77 ± 0.11 at 30 min, respectively ( p < 0.05). The biodistribution study showed that tumor uptake % ID per g (percentage of injected dose per gram of tissue) in the group of 68 Ga-DOTA-SD01 was 2.73 ± 0.34, and 1.77 ± 0.38 in the group of 68 Ga-DOTA-YSA; T/NT ratios (radioactivity of tumor to muscle) were 3.55 ± 0.12 and 3.05 ± 0.10 for both groups at 30 min, respectively ( p < 0.05). All these suggest that 68 Ga-DOTA-SD01 may act as a better novel PET imaging agent for EphA2 positive tumors, such as TNBC.

GRP78, a member of the HSP70 superfamily, is an endoplasmic reticulum chaperone protein overexpressed in various cancers, making it a promising target for cancer imaging and therapy. Positron emission tomography (PET) imaging offers unique advantages in real time, noninvasive tumor imaging, rendering it a suitable tool for targeting GRP78 in tumor imaging to guide targeted therapy. Several studies have reported successful tumor imaging using PET probes targeting GRP78. However, existing PET probes face challenges such as low tumor uptake, inadequate in vivo distribution, and high abdominal background signal. Therefore, this study introduces a novel peptide PET probe, [ 18 F]AlF-NOTA-c- D VAP, for targeted tumor imaging of GRP78. [ 18 F]AlF-NOTA-c- D VAP was radiolabeled with fluoride-18 using the aluminum-[ 18 F]fluoride ([ 18 F]AlF) method. The study assessed the partition coefficients, stability in vitro, and metabolic stability of [ 18 F]AlF-NOTA-c- D VAP. Micro-PET imaging, pharmacokinetic analysis, and biodistribution studies were carried out in tumor-bearing mice to evaluate the probe's performance. Docking studies and pharmacokinetic analyses of [ 18 F]AlF-NOTA-c- D VAP were also performed. Immunohistochemical and immunofluorescence analyses were conducted to confirm GRP78 expression in tumor tissues. The probe's binding affinity to GRP78 was analyzed by molecular docking simulation. [ 18 F]AlF-NOTA-c- D VAP was radiolabeled in just 25 min with a high yield of 51 ± 16%, a radiochemical purity of 99%, and molar activity within the range of 20-50 GBq/μmol. [ 18 F]AlF-NOTA-c- D VAP demonstrated high stability in vitro and in vivo, with a logD value of -3.41 ± 0.03. Dynamic PET imaging of [ 18 F]AlF-NOTA-c- D VAP in tumors showed rapid uptake and sustained retention, with minimal background uptake. Biodistribution studies revealed rapid blood clearance and excretion through the kidneys following a single-compartment reversible metabolic model. In PET imaging, the T/M ratios for A549 tumors (high GRP78 expression), MDA-MB-231 tumors (medium expression), and HepG2 tumors (low expression) at 60 min postintravenous injection were 10.48 ± 1.39, 6.25 ± 0.47, and 3.15 ± 1.15% ID/g, respectively, indicating a positive correlation with GRP78 expression. This study demonstrates the feasibility of using [ 18 F]AlF-NOTA-c- D VAP as a PET tracer for imaging GRP78 in tumors. The probe shows promising results in terms of stability, specificity, and tumor targeting. Further research may explore the clinical utility and potential therapeutic applications of this PET tracer for cancer diagnosis.

Introduction: Radiolabeled antibodies are promising tools for cancer diagnosis using nuclear medicine. A DOTA-chelating system is useful for preparing immuno-positron emission tomography and immuno-single-photon emission computed tomography probes with various radiometals. Radiolabeled antibodies are generally metabolized in the reticuloendothelial system, producing radiometabolites after proteolysis in hepatic lysosomes. Because of the bulkiness and extremely high hydrophilicity of DOTA, radiometabolites containing a radiometal-DOTA complex typically exhibit high and persistent localization in hepatic lysosomes. Radioactivity in the liver impairs the accurate diagnosis of cancer surrounding the liver and liver metastasis, and a high tumor/liver ratio is desirable. In this study, we reduced the hepatic radioactivity of radiometal-labeled antibodies containing a DOTA-chelating system. A cleavable linkage was inserted to liberate the radiometabolite, which exhibited a short residence time in hepatocytes., Methods: Using indium-111 ( 111 In)-labeled antibodies, we prepared 111 In-labeled galactosyl-neoglycoalbumins (NGAs) because they are useful for evaluating the residence time of radiometabolites in the liver. An 111 In-labeled NGA with a cleavable linkage ([ 111 In]In-DO3AiBu-Bn-FGK-NGA) was administered to normal mice, and biodistribution studies and metabolic analyses of urinary and fecal samples were performed with comparison to an 111 In-labeled NGA prepared by a conventional method ([ 111 In]In-DOTA-Bn-SCN-NGA). Then, 111 In-labeled antibodies ([ 111 In]In-DO3AiBu-Bn-FGK-IgG and [ 111 In]In-DOTA-Bn-SCN-IgG) were prepared using a procedure similar to that for 111 In-labeled NGAs. In vitro plasma stability and biodistribution were investigated for both 111 In-labeled antibodies in U87MG tumor-bearing mice., Results: Through the liberation of radiometabolites including [ 111 In]In-DO3AiBu-Bn-F, [ 111 In]In-DO3AiBu-Bn-FGK-NGA was cleared more rapidly from the liver than [ 111 In]In-DOTA-Bn-SCN-NGA (4.07 ± 1.54%ID VS 71.68 ± 3.03%ID at 6 h postinjection). [ 111 In]In-DO3AiBu-Bn-FGK-IgG exhibited lower tumor accumulation (8.83 ± 1.48%ID/g) but a significantly higher tumor/liver ratio (2.21 ± 0.53) than [ 111 In]In-DOTA-Bn-SCN-IgG (11.65 ± 2.17%ID/g in the tumor and a tumor/liver ratio of 0.85 ± 0.18) at 72 h after injection., Conclusion: A molecular design that reduces the high and persistent hepatic radioactivity of radiolabeled antibodies by liberating radiometabolites with a short hepatic residence time in lysosomes would be applicable for radiometal-labeled antibodies using a DOTA-chelating system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Noninvasive quantifying activated hepatic stellate cells (aHSCs) by molecular imaging is helpful for assessing disease progression and therapeutic responses of liver fibrosis. Our purpose is to develop platelet-derived growth factor receptor β (PDGFRβ)-targeted radioactive tracer for assessing liver fibrosis by positron emission tomography (PET) imaging of aHSCs., Methods: Comparative transcriptomics, immunofluorescence staining and flow cytometry were used to evaluate PDGFRβ as biomarker for human aHSCs and determine the correlation of PDGFRβ with the severity of liver fibrosis. The high affinity affibody for PDGFRβ (Z PDGFRβ ) was labeled with gallium-68 ( 68 Ga) for PET imaging of mice with carbon tetrachloride (CCl 4 )-induced liver fibrosis. Binding of the [ 68 Ga]Ga-labeled Z PDGFRβ ([ 68 Ga]Ga-DOTA-Z PDGFRβ ) for aHSCs in human liver tissues was measured by autoradiography., Results: PDGFRβ overexpressed in aHSCs was highly correlated with the severity of liver fibrosis in patients and CCl 4 -treated mice. The 68 Ga-labeled Z PDGFRβ affibody ([ 68 Ga]Ga-DOTA-Z PDGFRβ ) showed PDGFRβ-dependent binding to aHSCs. According to the PET imaging, hepatic uptake of [ 68 Ga]Ga-DOTA-Z PDGFRβ increased with the accumulation of aHSCs and collagens in the fibrotic livers of mice. In contrast, hepatic uptake of [ 68 Ga]Ga-DOTA-Z PDGFRβ decreased with spontaneous recovery or treatment of liver fibrosis, indicating that the progression and therapeutic responses of liver fibrosis in mice could be visualized by PDGFRβ-targeted PET imaging. [ 68 Ga]Ga-DOTA-Z PDGFRβ also bound human aHSCs and visualized fibrosis in patient-derived liver tissues., Conclusions: PDGFRβ is a reliable biomarker for both human and mouse aHSCs. PDGFRβ-targeted PET imaging could be used for noninvasive monitoring of liver fibrosis in mice and has great potential for clinical translation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

[Ln·DOTA] - complexes and systems derived therefrom are commonly used in MRI and optical bioimaging. These lanthanide(III) complexes are chiral, and, in solution, they are present in four forms, with two sets of enantiomers, with the ligand donors arranged in either a square antiprismatic, SAP, or twisted square antiprismatic geometry, TSAP. This complicated speciation is found in laboratory samples. To investigate speciation in biological media, when Ln·DOTA-like complexes interact with chiral biomolecules, six Eu·DOTA-monoamide complexes were prepared and investigated by using 1D and 2D 1 H NMR. To emulate the chirality of biological media, the amide pendant arm was modified with one or two chiral centers. It is known that a chiral center on the DOTA scaffold significantly influences the properties of the system. Here, it was found that chirality much further away from the metal center changes the available conformational space and that both chiral centers and amide cis / trans isomerism may need to be considered─a fact that, for the optically enriched materials, led to the conclusion that eight chemically different forms may need to be considered, instead of the four forms necessary for DOTA. The results reported here clearly demonstrate the diverse speciation that must be considered when correlating an observation to a structure of a lanthanide(III) complex.

Heteroarenes are ubiquitous motifs in bioactive molecules, conferring favourable physical properties when compared to their arene counterparts 1-3 . In particular, semisaturated heteroarenes possess attractive solubility properties and a higher fraction of sp 3 carbons, which can improve binding affinity and specificity. However, these desirable structures remain rare owing to limitations in current synthetic methods 4-6 . Indeed, semisaturated heterocycles are laboriously prepared by means of non-modular fit-for-purpose syntheses, which decrease throughput, limit chemical diversity and preclude their inclusion in many hit-to-lead campaigns 7-10 . Herein, we describe a more intuitive and modular couple-close approach to build semisaturated ring systems from dual radical precursors. This platform merges metallaphotoredox C(sp 2 )-C(sp 3 ) cross-coupling with intramolecular Minisci-type radical cyclization to fuse abundant heteroaryl halides with simple bifunctional feedstocks, which serve as the diradical synthons, to rapidly assemble a variety of spirocyclic, bridged and substituted saturated ring types that would be extremely difficult to make by conventional methods. The broad availability of the requisite feedstock materials allows sampling of regions of underexplored chemical space. Reagent-controlled radical generation leads to a highly regioselective and stereospecific annulation that can be used for the late-stage functionalization of pharmaceutical scaffolds, replacing lengthy de novo syntheses., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

While most FDA-approved peptide drugs are cyclic, robust cyclization chemistry of peptides and the deconvolution of the cyclic peptide sequences using tandem mass spectrometry render cyclic peptide drug discovery difficult. In this chapter, the protocol for the successful synthesis of tetrazine-linked cyclic peptide library in solid phase, which shows both robust cyclization and easy sequence deconvolution, is described. The protocol for the linearization and cleavage of cyclic peptides from the solid phase by simple UV light irradiation, followed by accurate sequencing using tandem mass spectrometry, is described. We describe the troubleshooting for this dithiol bis-arylation reaction and for the successful cleavage of the aryl cyclic peptide into linear form. This method for efficient solid-phase macrocyclization can be used for the rapid production of loop-based peptides and screening for inhibition of protein-protein interactions, by using the covalent inverse electron-demand Diels Alder reaction to supplement the non-covalent interaction between a protein and its peptide binder, isolating highly selective peptides in the process., (Copyright © 2024. Published by Elsevier Inc.)

Background: Prostate-specific membrane antigen (PSMA) is an ideal target for molecular imaging and targeted radionuclide therapy in prostate cancer. Consequently, various PSMA ligands were developed. Some of these molecules are functionalized with a chelator that can host radiometals, such as 68 Ga for PET imaging. The 68 Ga radiolabeling step benefits from process automation, making it more robust and reducing radiation exposure., Objective: To design a single automated radiolabeling protocol for the GMP-compliant preparation of [ 68 Ga]Ga-PSMA-11, transposable to the production of [ 68 Ga]Ga-PSMA-617 and [ 68 Ga]Ga-PSMA-I&T., Methods: A GAIA ® synthesis module and a GALLIAD ® generator were used. Radio-TLC and radio-HPLC methods were validated for radiochemical purity (RCP) determination. Three [ 68 Ga]Ga-PSMA-11 validation batches were produced and thoroughly tested for appearance and pH, radionuclide identity and purity, RCP, stability, residual solvent and sterility. Minimal modifications were made to the reagents and disposables for optimal application to other PSMA ligands., Results: [ 68 Ga]Ga-PSMA-11 for clinical application was produced in 27 min. The 3 validation batches met the quality criteria expected by the European Pharmacopoeia to allow routine production. For optimal transposition to PSMA-617, the solid phase extraction cartridge was changed to improve purification of the radiolabeled product. For application to PSMA-I&T, the buffer solution initially used was replaced by HEPES 2.7 M to achieve good radiochemical yields. Residual HEPES content was checked in the final product and was below the Ph. Eur. threshold., Conclusion: A single automated radiolabeling method on the GAIA ® module was developed and implemented for 68 Ga radiolabeling of 3 PSMA ligands, with slight adjustments for each molecule., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The human prostate-specific membrane antigen (PSMA) is substantially up-regulated in metastatic prostate cancer (PCa) cells. PSMA can be targeted by 177 Lu conjugated to PSMA-617, a high-affinity ligand for the PSMA. The binding of the radioligand, 177 Lu-PSMA-617, results in its internalisation and delivery of β-radiation into the cancer cells. However, PSMA-617, a component of the final product in the synthesis of the radioligand, may also play a role in the pathophysiology of PCa cells. The present study aimed to clarify the effects of PSMA-617 (10, 50 and 100 nM) on the expression of PSMA in PSMA-positive LNCaP cells, their proliferation, 177 Lu-PSMA-617-induced cell death by WST-1 and lactate dehydrogenase assays, immunohistochemistry, western blotting, immunofluorescence staining and uptake of 177 Lu-PSMA-617. PSMA-617 at 100 nM concentration induced cell-growth arrest, down-regulated cyclin D1 and cyclin E1 (by 43 and 36%, respectively) and up-regulated the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 (by 48%). Immunofluorescence staining demonstrated reduced content of DNA, pointing to a lower rate of cell division. PSMA-617 (up to 100 nM) did not alter the uptake of 177 Lu-PSMA-617 into the LNCaP cells. Interestingly, simultaneous treatment with 177 Lu-PSMA-617 and PSMA-617 for 24 and 48 h substantially potentiated the cell-death promoting effects of the radioligand. In conclusion, the combination of impeding tumour cell proliferation by PSMA-617 and its potentiation of the radiation-induced cell death brought about by 177 Lu-PSMA-617 in PCa cells may considerably improve the outcome of the radiation therapy with 177 Lu-PSMA-617, especially in patients with decreased radiosensitivity of PCa cells to the radioligand., (© 2023. The Author(s).)

Purpose: A novel HER2 affibody-based molecular probe, [ 18 F]AlF-RESCA-HER2-BCH, was developed for reducing renal uptake, evaluated, and compared with [ 18 F]AlF-NOTA-HER2-BCH., Methods: In preclinical studies, micro-PET/CT was performed using HER2-positive gastric cancer patient-derived xenografts (PDX) model at 0.5-1 (dynamic), 2, 4, and 6 h post-injection. For blocking experiment, 0.5 mg cold affibody was co-injected with probes. Biodistribution were performed on HER2-positive PDX models at 2 h post-injection. For clinical study, PET/CT images were acquired at 2 h and 4 h after injection of 231.29 ± 17.77 MBq [ 18 F]AlF-NOTA-HER2-BCH or [ 18 F]AlF-RESCA-HER2-BCH in five breast cancer patients (4 HER2-positive and 1 HER2-low). Standardized uptake values (SUVs) were measured in tumors and source-organs for semi-quantitative analysis. The OLINDA/EXM software (version 1.2) was used to calculate the radiation doses., Results: [ 18 F]AlF-NOTA-HER2-BCH and [ 18 F]AlF-RESCA-HER2-BCH were stably labeled with [ 18 F]F, with high binding specificity and affinity to HER2. Micro-PET/CT of both tracers could clearly visualize HER2-positive PDX tumors with high uptake of 16.24 ± 1.74% ID/g and 14.39 ± 2.45% ID/g at 2 h post-injection. The renal accumulation of [ 18 F]AlF-RESCA-HER2-BCH was significantly lower than that of [ 18 F]AlF-NOTA-HER2-BCH (5.16 ± 0.22% ID/g vs. 158.73 ± 5.44% ID/g at 2 h, p < 0.0001). In the clinical study, both [ 18 F]AlF-NOTA-HER2-BCH and [ 18 F]AlF-RESCA-HER2-BCH demonstrated favorable tumor targeting and image contrast. [ 18 F]AlF-RESCA-HER2-BCH showed a higher SUVmax in both primary tumor and metastases, and a significantly higher target-to-nontarget ratio in metastases than [ 18 F]AlF-NOTA-HER2-BCH. Moreover, [ 18 F]AlF-RESCA-HER2-BCH had lower renal accumulation (43.56 ± 7.88 vs. 79.81 ± 3.81 at 2 h, p < 0.0001; 33.23 ± 6.89 vs. 78.63 ± 4.00 at 4 h, p < 0.0001) as well as a significantly lower renal absorbed dose than [ 18 F]AlF-NOTA-HER2-BCH (0.4450 ± 0.1117 mGy/MBq vs. 0.8030 ± 0.1604 mGy/MBq, p < 0.01)., Conclusions: [ 18 F]AlF-RESCA-HER2-BCH tended to provide better image contrast than [ 18 F]AlF-NOTA-HER2-BCH with a higher target-to-nontarget ratio in detection of metastases. Notably, [ 18 F]AlF-RESCA-HER2-BCH had lower renal accumulation than [ 18 F]AlF-NOTA-HER2-BCH., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study addresses the question whether inductively coupled plasma mass spectrometry (ICP-MS) can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals. In a "proof-of-concept" study, the prostate-specific membrane antigen (PSMA)-targeting [ 175 Lu]Lu-PSMA-617 and [ 159 Tb]Tb-PSMA-617 were compared with their respective radiolabeled analogues, [ 177 Lu]Lu-PSMA-617 (PLUVICTO, Novartis) and [ 161 Tb]Tb-PSMA-617. ICP-MS and conventional γ-counting of the cell samples revealed almost identical results (<6% absolute difference between the two technologies) for the in vitro uptake and internalization of the (radio)metal conjugates, irrespective of the employed methodology. In vivo, an equal uptake in PSMA-positive PC-3 PIP tumor xenografts was determined 1 h after the injection of [ 175 Lu]Lu-/[ 177 Lu]Lu-PSMA-617 (41 ± 6% ID/g and 44 ± 12% IA/g, respectively) and [ 159 Tb]Tb-/[ 161 Tb]Tb-PSMA-617 (44 ± 5% ID/g and 44 ± 5% IA/g, respectively). It was further revealed that it is crucial to use the same ratios of the (radio)metal-labeled and unlabeled ligands for both methodologies to obtain equal data in organs in which receptor saturation was reached such as the kidneys (12 ± 2% ID/g vs 10 ± 1% IA/g, 1 h after injection). The data of this study demonstrate that the use of high-sensitivity ICP-MS allows reliable and predictive quantification of compounds labeled with stable metal isotopes in cell and tissue samples obtained in preclinical studies. It can, hence, be employed as a valid alternative to the state-of-the-art γ-counting methodology to detect radioactive ligands.

Erlotinib is a first generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) which was granted Food and Drug administration (FDA) approval for treatment of patients with locally advanced or metastatic NSCLC. The present study aimed at development of radiolabeled erlotinib variants as tyrosine kinase inhibitors. Three DOTA-erlotinib conjugates were prepared for radiolabeling with 177 Lu. The terminal alkyne group of erlotinib was modified by performing Cu-catalyzed click chemistry and three different linkers were introduced which were then conjugated to the chelator, DOTA. The DOTA-erlotinib conjugates were characterized by 1 H NMR and ESI-MS. 177 Lu-DOTA-erlotinib complexes were characterized using nat Lu-DOTA-erlotinib conjugates. The 177 Lu-complexes exhibited high in vitro stability in human serum up to 48 h. They were highly hydrophilic in nature as observed from their log P o/w values ( 177 Lu-DOTA-propyl-Er: -2.5 ± 0.1; 177 Lu-DOTA-PEG 3 -Er: -3.0 ± 0.1; 177 Lu-DOTA-PEG 6 -Er: -3.3 ± 0.1). The MTT assay in A431 human epidermoid carcinoma cell lines indicates that the chemical modification at the terminal alkyne group of the erlotinib molecule does not have significant effect on its TKI property. Biodistribution studies in normal Swiss mice demonstrated fast clearance and excretion of 177 Lu-labeled erlotinib complexes. These studies indicate that erlotinib variants with hydrophobic pharmacokinetic modifiers/chelators may enhance the retention of 177 Lu-labeled complexes in blood thereby increasing the probability to reach EGFR-expressing tumor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)