Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.

The expression level of PD-L1 in tumor tissue is considered one of the effective biomarkers to guide PD-1/PD-L1 therapy. Quantifying whole-body PD-L1 expression by SPECT imaging may help in selecting patients that potentially respond to PD-1/PD-L1 therapy. Nanobody is the smallest antibody fragment with antigen-binding ability that is well suited for radionuclide imaging. Nevertheless, high retention of radioactivity in the kidney may limit its clinical translation. The present study aimed to screen, design, and prepare a nanobody-based SPECT probe with rapid renal clearance to evaluate the PD-L1 expression level in vivo noninvasively. A phage library was constructed by immunizing alpaca with recombinant human PD-L1 protein, and 17 anti-PD-L1 nanobodies were screened by the phage display technique. After sequence alignment and flow cytometry analysis, APN09 was selected as the candidate nanobody, and a GGGC chelator was attached to its C-terminus for 99mTc labeling to prepare a SPECT imaging probe. The affinity and specificity of 99mTc-APN09 were evaluated by protein and cell-binding experiments, and SPECT imaging and biodistribution were performed in a mouse model with bilateral transplantation of A549 and A549PD-L1 tumors. The ability of 99mTc-APN09 to quantify the PD-L1 expression level in vivo was validated in tumor models with different PD-L1 expression levels. 99mTc-APN09 had a radiochemical purity higher than 99% and a binding equilibrium dissociation constant of 21.44 ± 1.65 nM with hPD-L1, showing high affinity. SPECT imaging results showed that 99mTc-APN09 could efficiently detect PD-L1-positive tumors within 0.5 h, and the quantitative results of SPECT were well correlated with the expression level of PD-L1 in cell lines. SPECT imaging and biodistribution results also showed that 99mTc-APN09 was rapidly cleared from the kidney in 2 h postinjection. 99mTc-APN09 was a simple and stable tool for visualizing PD-L1 expression in the whole body. In addition, due to its significant reduction in renal retention, it has better prospects for clinical translation.

Construction and Preclinical Evaluation of 124I/125I-Labeled Antibody Targeting T Cell Immunoglobulin and Mucin Domain-3.

T cell immunoglobulin and mucin domain-3 (TIM3; HAVCR2) is a transmembrane protein that exerts negative regulatory control over T cell responses. Studies have demonstrated an upregulation of TIM3 expression in tumor-infiltrating lymphocytes (TILs) in cancer patients. In this investigation, a series of monoclonal antibodies targeting TIM3 were produced by hybridoma technology. Among them, C23 exhibited favorable biological properties. To enable specific binding, we developed a 124I/125I-C23 radio-tracer via N-bromosuccinimide (NBS)-mediated labeling of the monoclonal antibody C23. Binding affinity and specificity were assessed using the 293T-TIM3 cell line, which overexpresses TIM3, and the parent 293T cells. Furthermore, biodistribution and in vivo imaging of 124I/125I-C23 were examined in HEK293TIM3 xenograft models and allograft models of 4T1 (mouse breast cancer cells) and CT26 (mouse colon cancer cells). Micro-PET/CT imaging was conducted at intervals of 4, 24, 48, 72, and/or 96 h post intravenous administration of 3.7-7.4 MBq 124I-C23 in the respective model mice. Additionally, immunohistochemistry (IHC) staining of TIM3 expression in dissected tumor organs was performed, along with an assessment of the corresponding expression of Programmed Death 1 (PD1), CD3, and CD8 in the tumors. The C23 monoclonal antibody (mAb) specifically binds to TIM3 protein with a dissociation constant of 23.28 nM. The 124I-C23 and 125I-C23 radio-tracer were successfully prepared with a labeling yield of 83.59 ± 0.35% and 92.35 ± 0.20%, respectively, and over 95.00% radiochemical purity. Stability results indicated that the radiochemical purity of 124I/125I-C23 in phosphate-buffered saline (PBS) and 5% human serum albumin (HSA) was still >80% after 96 h. 125I-C23 uptake in 293T-TIM3 cells was 2.80 ± 0.12%, which was significantly higher than that in 293T cells (1.08 ± 0.08%), and 125I-C23 uptake by 293T-TIM3 cells was significantly blocked at 60 and 120 min in the blocking groups. Pharmacokinetics analysis in vivo revealed an elimination time of 14.62 h and a distribution time of 0.4672 h for 125I-C23. Micro-PET/CT imaging showed that the 124I-C23 probe uptake in the 293T-TIM3 model significantly differed from that of the negative control group and blocking group. In the humanized mouse model, the 124I-C23 probe had obvious specific uptake in the 4T1 and CT26 models and maximum uptake at 24 h in tumor tissues (SUVmax (the maximum standardized uptake value) in 4T1 and CT26 humanized TIM3 murine tumor models: 0.59 ± 0.01 and 0.76 ± 0.02, respectively). Immunohistochemistry of tumor tissues from these mouse models showed comparable TIM3 expression. CD3 and CD8 cells and PD-1 expression were also observed in TIM3-expressing tumor tissues. The TIM3-targeting antibody C23 showed good affinity and specificity. The 124I/125I-C23 probe has obvious targeting specificity for TIM3 in vitro and in vivo. Our results suggest that 124I/125I-C23 is a promising tracer for TIM3 imaging and may have great potential in monitoring immune checkpoint drug efficacy.

124I-labeled anti-CD147 antibody for noninvasive detection of CD147-positive pan-cancers: construction and preclinical studies.

Extracellular matrix metalloproteinase inducer CD147 is a glycoprotein on the cell surface. There is minimal expression of CD147 in normal epithelial and fetal tissues, but it is highly expressed in a number of aggressive tumors. CD147 has been implicated in pan-cancer immunity and progression. With the development of CD147-targeting therapeutic strategy, accurate detection of CD147 expression in tumors and its changes during the therapy is necessary. In this study we constructed a novel radiotracer by labeling the anti-CD147 mAb with radionuclide 124/125I (124/125I-anti-CD147) for noninvasive detection of CD147 expression in pan-cancers, and characterized its physicochemical properties, affinity, metabolic characteristics, biodistribution and immunoPET imaging with 124I-IgG and 18F-FDG as controls. By examining the expression of CD147 in cancer cell lines, we found high CD147 expression in colon cancer cells LS174T, FADU human pharyngeal squamous cancer cells and 22RV1 human prostate cancer cells, and low expression of CD147 in human pancreatic cancer cells ASPC1 and human gastric cancer cells BGC823. 124/125I-anti-CD147 was prepared using N-bromine succinimide (NBS) as oxidant and purified by PD-10 column. Its radiochemical purity (RCP) was over 99% and maintained over 85% in saline or 5% human serum albumin (HSA) for more than 7 d; the RCP of 125I-anti-CD147 in blood was over 90% at 3 h post injection (p.i.) in healthy mice. The Kd value of 125I-anti-CD147 to CD147 protein was 6.344 nM, while that of 125I-IgG was over 100 nM. 125I-anti-CD147 showed much greater uptake in CD147 high-expression cancer cells compared to CD147 low-expression cancer cells. After intravenous injection in healthy mice, 125I-anti-CD147 showed high initial uptake in blood pool and liver, the uptake was decreased with time. The biological half-life of distribution and clearance phases in healthy mice were 0.63 h and 19.60 h, respectively. The effective dose of 124I-anti-CD147 was estimated as 0.104 mSv/MBq. We conducted immunoPET imaging in tumor-bearing mice, and demonstrated a significantly higher tumor-to-muscle ratio of 124I-anti-CD147 compared to that of 124I-IgG and 18F-FDG in CD147 (+) tumors. The expression levels of CD147 in cells and tumors were positively correlated with the maximum standardized uptake value (SUVmax) (P < 0.01). In conclusion, 124/125I-anti-CD147 displays high affinity to CD147, and represents potential for the imaging of CD147-positive tumors. The development of 124I-anti-CD147 may provide new insights into the regulation of tumor microenvironment and formulation of precision diagnosis and treatment programs for tumors.

Oxidative stress-induced temporal activation of ERK1/2 phosphorylates coreceptor of Wnt/ß-catenin for myofibroblast formation in human lens epithelial cells.

Purpose: Posterior capsular opacification (PCO) is the most common complication postcataract surgery, and its underlying mechanisms involve epithelial-mesenchymal transition (EMT) of remnant lens epithelial cells (LECs) in response to drastic changes in stimuli in the intraocular environment, such as oxidative stress and growth factors. Wnt/ß-catenin signaling is a major pathway mediating oxidative stress-induced EMT in LECs, but its interplay with other transduction pathways remains little known in the development of PCO. ERK1/2 signaling is the downstream component of a phosphorelay pathway in response to extracellular stimuli (e.g., reactive oxygen species), and its activation regulates multiple cellular processes, including proliferation and EMT. Thus, this study aimed to investigate how ERK1/2 signaling and Wnt/ß-catenin pathway crosstalk in oxidative stress-induced EMT in LECs. Methods: Hydrogen peroxide (H2O2) at 50 µM treatment for 48 h was used to establish a moderate oxidative stress-induced EMT model in LECs. ERK1/2 signaling was inhibited using MEK1/2 inhibitor U0126 at 20 µM. Western blotting was used to quantify protein expression of various biomarkers of EMT and phosphorylated components in ERK1/2 and Wnt/ß-catenin signaling. LEC proliferation was determined using an EdU staining assay and expression of proliferating cellular nuclear antigen (PCNA). Subcellular localization of biomarker proteins was visualized with immunofluorescent staining. Results: Under the moderate level of H2O2-induced EMT in LECs, ERK1/2 signaling was activated, as evidenced by a marked increase in the ratio of phosphorylated ERK1/2 to total ERK1/2 at early (i.e., 5-15 min) and late time points (i.e., 12 h); the canonical Wnt/ß-catenin pathway was activated by H2O2 at 48 h. LECs exposed to H2O2 exhibited hyperproliferation and EMT; however, these were restored by inhibition of ERK1/2 signaling demonstrated by reduced DNA synthesis and PCNA expression for cellular proliferation and altered expression of various EMT protein markers, including E-cadherin, α-SMA, and vimentin. More importantly, inhibition of ERK1/2 signaling reduced ß-catenin accumulation in the activated Wnt/ß-catenin signaling cascade. Specifically, there was significant downregulation in the phosphorylation level of LRP6 at Ser 1490 and GSK-3ß at Ser 9, the key coreceptor of Wnt and regulator of ß-catenin, respectively. Conclusions: ERK1/2 signaling plays a crucial role in the moderate level of oxidative stress-induced EMT in LECs. Pharmacologically blocking ERK1/2 signaling significantly inhibited LEC proliferation and EMT. Mechanistically, ERK1/2 signaling regulated Wnt/ß-catenin cascade by phosphorylating Wnt coreceptor LRP6 at Ser 1490 in the plasma membrane. These results shed light on a potential molecular switch of ERK1/2 and Wnt/ß-catenin crosstalk underlying the development of PCO.

Immuno-PET of colorectal cancer with a CEA-targeted [68 Ga]Ga-nanobody: from bench to bedside.

PURPOSE: An accurate diagnosis of colorectal carcinoma (CRC) can assist physicians in developing reasonable therapeutic regimens, thereby significantly improving the patient's prognosis. Carcinoembryonic antigen (CEA)-targeted PET imaging shows great potential for this purpose. Despite showing remarkable abilities to detect primary and metastatic CRC, previously reported CEA-specific antibody radiotracers or pretargeted imaging are not suitable for clinical use due to poor pharmacokinetics and complicated imaging procedures. In contrast, radiolabeled nanobodies exhibit ideal characteristics for PET imaging, for instance, rapid clearance rates and excellent distribution profiles, allowing same-day imaging with sufficient contrast. In this study, we developed a novel CEA-targeted nanobody radiotracer, [68 Ga]Ga-HNI01, and assessed its tumor imaging ability and biodistribution profile in preclinical xenografts and patients with primary and metastatic CRC. METHODS: The novel nanobody HNI01 was acquired by immunizing the llama with CEA proteins. [68 Ga]Ga-HNI01 was synthesized by site-specifically conjugating [68 Ga]Ga with tris(hydroxypyridinone) (THP). Small-animal PET imaging and biodistribution studies were performed in CEA-overexpressed LS174T and CEA-low-expressed HT-29 tumor models. Following successful preclinical assessment, a phase I study was conducted on 9 patients with primary and metastatic CRC. Study participants received 151.21 ± 25.25 MBq of intravenous [68 Ga]Ga-HNI01 and underwent PET/CT scans at 1 h and 2 h post injection. Patients 01-03 also underwent whole-body dynamic PET imaging within 0-40 min p.i. All patients underwent [18F]F-FDG PET/CT imaging within 1 week after [68 Ga]Ga-HNI01 imaging. Tracer distribution, pharmacokinetics, and radiation dosimetry were calculated. RESULTS: [68 Ga]Ga-HNI01 was successfully synthesized within 10 min under mild conditions, and the radiochemical purity was more than 98% without purification. Micro-PET imaging with [68 Ga]Ga-HNI01 revealed clear visualization of LS174T tumors, while signals from HT-29 tumors were significantly lower. Biodistribution studies indicated that uptake of [68 Ga]Ga-HNI01 in LS174T and HT-29 was 8.83 ± 3.02%ID/g and 1.81 ± 0.87%ID/g, respectively, at 2 h p.i. No adverse events occurred in all clinical participants after the injection of [68 Ga]Ga-HNI01. A fast blood clearance and low background uptake were observed, and CRC lesions could be visualized with high contrast as early as 30 min after injection. [68 Ga]Ga-HNI01 PET could clearly detect metastatic lesions in the liver, lung, and pancreas and showed superior ability in detecting small metastases. A significant accumulation of radioactivity was observed in the kidney, and normal tissues physiologically expressing CEA receptors showed slight uptakes of [68 Ga]Ga-HNI01. An interesting finding was that strong uptake of [68 Ga]Ga-HNI01 was found in non-malignant colorectal tissues adjacent to the primary tumor in some patients, suggesting abnormal CEA expression in these healthy tissues. CONCLUSION: [68 Ga]Ga-HNI01 is a novel CEA-targeted PET imaging radiotracer with excellent pharmacokinetics and favorable dosimetry profiles. [68 Ga]Ga-HNI01 PET is an effective and convenient imaging tool for detecting CRC lesions, particularly for identifying small metastases. Furthermore, its high specificity for CEA in vivo makes it an ideal tool for selecting patients for anti-CEA therapy.

High in-vivo stability in preclinical and first-in-human experiments with [18F]AlF-RESCA-MIRC213: a 18F-labeled nanobody as PET radiotracer for diagnosis of HER2-positive cancers.

PURPOSE: [18F]AlF-RESCA was introduced as a core particularly useful for 18F-labeling of heat-sensitive biomolecules. However, no translational studies have been reported up to now. Herein, we reported the first-in-human evaluation of an 18F-labeled anti-HER2 nanobody MIRC213 as a PET radiotracer for imaging HER2-positive cancers. METHODS: MIRC213 was produced by E. coli and conjugated with ( ±)-H3RESCA-Mal. [18F]AlF-RESCA-MIRC213 was prepared at room temperature. Its radiochemical purity and stability of were determined by radio-HPLC with the size-exclusion chromatographic column. Cell uptake was performed in NCI-N87 (HER2 +) and MCF-7 (HER2-) cells and the cell-binding affinity was verified in SK-OV-3 (HER2 +) cells. Small-animal PET/CT was performed using SK-OV-3, NCI-N87, and MCF-7 tumor-bearing mice at 30 min, 1 h, and 2 h post-injection. For blocking experiment, excess MIRC213 was co-injected with radiotracer. Biodistribution were performed on SKOV-3 and MCF-7 tumor-bearing mice at 2 h post-injection. For clinical study, PET/CT images were acquired at 2 h and 4 h after injection of [18F]AlF-RESCA-MIRC213 (1.85-3.7 MBq/kg) in six breast cancer patients (3 HER2-positive and 3 HER2-negative). All patients underwent [18F]-FDG PET/CT within a week for tissue selection purpose. Distribution and dosimetry were calculated. Standardized uptake values (SUV) were measured in tumors and normal organs. RESULTS: MIRC213 was produced with > 95% purity and modified with RESCA to obtain RESCA-MIRC213. [18F]AlF-RESCA-MIRC213 was prepared within 20 min at room temperature with the radiochemical yield of 50.48 ± 7.6% and radiochemical purity of > 98% (n > 10), and remained stable in both PBS (88%) and 5% HSA (92%) after 6 h. The 2 h cellular uptake of [18F]AlF-RESCA-MIRC213 in NCI-N87 cells was 11.22 ± 0.60 AD%/105 cells. Its binding affinity Kd value was determined to be 1.23 ± 0.58 nM. Small-animal PET/CT with [18F]AlF-RESCA-MIRC213 can clearly differentiate SK-OV-3 and NCI-N87 tumors from MCF-7 tumors and background with a high uptake of 4.73 ± 1.18 ID%/g and substantially reduced to 1.70 ± 0.13 ID%/g for the blocking group (p < 0.05) in SK-OV-3 tumors at 2 h post-injection. No significant bone radioactivity was seen in the tumor-bearing animals. In all six breast cancer patients, there was no adverse reaction during study. The uptake of [18F]AlF-RESCA-MIRC213 was mainly in lacrimal gland, parotid gland, submandibular gland, thyroid gland, gallbladder, kidneys, liver, and intestines. There was no significant bone radioactivity accumulation in cancer patients. [18F]AlF-RESCA-MIRC213 had significantly higher tumor uptake in lesions from HER2-positive patients than that lesions from HER2-negative patients (SUVmax of 3.62 ± 1.56 vs. 1.41 ± 0.41, p = 0.0012) at 2 h post-injection. The kidneys received the highest radiation dose of 2.42 × 10-1 mGy/MBq, and the effective dose was 1.56 × 10-2 mSv/MBq. CONCLUSIONS: [18F]AlF-RESCA-MIRC213 could be prepared with high radiolabeling yield under mild conditions. [18F]AlF-RESCA-MIRC213 has relatively high stability both in vitro and in vivo. The results from clinical transformation suggest that [18F]AlF-RESCA-MIRC213 PET/CT is a safe procedure with favorable pharmacokinetics and dosimetry profile, and it is a promising new PET radiotracer for noninvasive diagnosis of HER2-positive cancers.

Preclinical evaluation and pilot clinical study of [68Ga]Ga-THP-APN09, a novel PD-L1 targeted nanobody radiotracer for rapid one-step radiolabeling and PET imaging.

PURPOSE: Programmed cell death protein-1/ligand-1 (PD-1/L1) blockade has been a breakthrough in the treatment of patients with non-small cell lung cancer (NSCLC), but there is still a lack of effective methods to screen patients. Here we report a novel 68 Ga-labeled nanobody [68 Ga]Ga-THP-APN09 for PET imaging of PD-L1 status in mouse models and a first-in-human study in NSCLC patients. METHODS: [68 Ga]Ga-THP-APN09 was prepared by site-specific radiolabeling, with no further purification. Cell uptake assays were completed in the human lung adenocarcinoma cell line A549, NSCLC cell line H1975 and human PD-L1 gene-transfected A549 cells (A549PD-L1). The imaging to image PD-L1 status and biodistribution were investigated in tumor-bearing mice of these three tumor cell types. The first-in-human clinical translational trial was registered as NCT05156515. The safety, radiation dosimetry, biodistribution, and correlations of tracer uptake with immunohistochemical staining and major pathologic response (MPR) were evaluated in NSCLC patients who underwent adjuvant immunotherapy combined with chemotherapy. RESULTS: Radiosynthesis of [68 Ga]Ga-THP-APN09 was achieved at room temperature and a pH of 6.0-6.5 in 10 min with a high radiochemical yield (> 99%) and 13.9-27.8 GBq/µmol molar activity. The results of the cell uptake study reflected variable levels of surface PD-L1 expression observed by flow cytometry in the order A549PD-L1 > H1975 > A549. In small-animal PET/CT imaging, H1975 and A549PD-L1 tumors were clearly visualized in an 8.3:1 and 2.2:1 ratios over PD-L1-negative A549 tumors. Ex vivo biodistribution studies showed that tumor uptake was consistent with the PET results, with the highest A549PD-L1 being taken up the most (8.20 ± 0.87%ID/g), followed by H1975 (3.69 ± 0.50%ID/g) and A549 (0.90 ± 0.16%ID/g). Nine resectable NSCLC patients were enrolled in the clinical study. Uptake of [68 Ga]Ga-THP-APN09 was mainly observed in the kidneys and spleen, followed by low uptake in bone marrow. The radiation dose is within a reliable range. Tumor uptake was positively correlated with PD-L1 expression TPS (rs = 0.8763, P = 0.019). Tumor uptake of [68 Ga]Ga-THP-APN09 (SUVmax) in MPR patients was higher than that in non-MPR patients (median SUVmax 2.73 vs. 2.10, P = 0.036, determined with Mann-Whitney U-test). CONCLUSION: [68 Ga]Ga-THP-APN09 has the potential to be transformed into a kit-based radiotracer for rapid, simple, one-step, room temperature radiolabeling. The tracer can detect PD-L1 expression levels in tumors, and it may make it possibility to predict the response of PD-1 immunotherapy combined with chemotherapy. Confirmation in a large number of cases is needed. TRIAL REGISTRATION: Clinical Trial (NCT05156515). Registered 12 December 2021.

Construction and Preclinical Evaluation of a 124/125I-Labeled Specific Antibody Targeting PD-L2 in Lung Cancer.

Programmed cell death-ligand 2 (PD-L2) is an important emerging molecule of the immune checkpoint, which is closely related to the prognosis of patients with immune checkpoint inhibitor (ICI) therapy. The quantification of PD-L2 can provide a potential reference for patients who benefit from ICI treatment. In this study, we used iodine isotope (nat/124/125I)-labeled PD-L2 antibody (ATL2) to noninvasively detect PD-L2 expression in mice with human lung adenocarcinoma A549 cell lines. The radiochemical yields of 125I-ATL2 and 124I-ATL2 were 73.56 ± 3.72% and 69.46 ± 2.05%, respectively. The radiochemical purity (RCP) of the tracers was more than 99%. The positive cell line A549-PDL2 was constructed by lentivirus. Western blot, immunofluorescence, and flow cytometry indicated that the A549-PDL2 cells showed a higher PD-L2 protein level than the A549 cells. The dissociation constant of 125I-ATL2 binding to the PD-L2 protein was 7.25 nM. Cellular uptake experiments confirmed that the uptake of 125I-ATL2 in A549-PDL2 cells was higher than that in A549 cells at each time point (P < 0.0001). Micro-PET/CT showed significant uptake in the tumor region of A549-PDL2 tumor-bearing mice 24 h postinjection of 124I-ATL2 compared with that of other groups (SUVmax = 0.75 ± 0.06, 0.19 ± 0.07, and 0.27 ± 0.05, respectively). Consistently, the biodistribution of the tracers at 24 h postinjection showed a higher tumor uptake in A549-PDL2 mice (7.11 ± 0.38 %ID/g for 124I-ATL2 in A549-PDL2 mice vs 2.72 ± 0.15 %ID/g for 124I-ATL2 in A549 mice vs 3.89 ± 0.65 %ID/g for 124I-IgG in A549-PDL2 mice). The dosimetry estimation by using Olinda software showed that the effective dose of 124I-ATL2 was 3.62 × 10-2 mSv/MBq, which is within the range of acceptable doses. Immunohistochemical results further confirmed that the expression of PD-L2 in the tumor tissues of A549-PDL2-bearing mice was higher than that of the A549 model mice. In conclusion, the development of 124/125I-ATL2 provides the first noninvasive quantification of PD-L2 expression in lung cancer by molecular imaging, which provides a new reference for screening potential beneficiaries of ICI therapy.

IgG-Binding Nanobody Capable of Prolonging Nanobody-Based Radiotracer Plasma Half-Life and Enhancing the Efficacy of Tumor-Targeted Radionuclide Therapy.

Nanobodies have been developed rapidly as targeted probes for molecular imaging owing to their high affinity, outstanding tissue penetration, and rapid blood clearance. However, the short retention time at the tumor site limits their application in targeted radionuclide therapy. In this study, we designed a dual-targeting nanobody referred to as MIRC213-709, which can specifically bind to the HER2 receptor in tumor cell lines with high affinity (by nanobody MIRC213) and endogenous IgG in plasma to prolong the half-life by the MIRC213 C-terminal fusion nanobody, MIRC709. The nanobodies were site-specifically radiolabeled with 99mTc and 177Lu, and radiochemical purity was >95% after purification. The long blood circulation time and tumor retention property of 99mTc/177Lu-MIRC213-709 were confirmed by a blood clearance assay, single-photon emission computed tomography (SPECT), and a biodistribution study. The blood clearance assay showed that the distribution phase half-life (T1/2α) and elimination phase half-life (T1/2ß) of 99mTc-MIRC213-709 were 6.74- and 19.04-fold longer than those of 99mTc-MIRC213, respectively. The SPECT/CT and biodistribution results showed that the highest uptake of 177Lu-MIRC213 in the NCI-N87 model was 5.24 ± 0.95% ID/g at 6 h p.i., while the highest uptake of 177Lu-MIRC213-709 in the NCI-N87 model was 30.82 ± 7.29% ID/g at 48 h p.i. Compared with 177Lu-MIRC213, 177Lu-MIRC213-709 had a 16.9-fold increased tumor cumulative uptake (2606 ± 195.1 vs 153.9 ± 22.37% ID/g·h). The targeted radionuclide therapy assay was performed in the NCI-N87 tumor model, and treatment monitoring ended on day 32. The post-treatment/pretreatment tumor volumes were 12.99 ± 1.66, 3.58 ± 0.96, 1.26 ± 0.17, and 1.54 ± 0.50 in the 0, 9, and 18 MBq single-dose groups and the two 9 MBq divided dose group (14 days apart), respectively. All treatment groups showed significant therapeutic effects (P < 0.0001). Thus, fusion with the IgG-binding nanobody MIRC709 provides MIRC213 derivatives with improved metabolic properties for targeted radionuclide therapy.

MnSO4-promoted S-O bond cleavage for synthesizing functionalized sulfonium ylides from activated alkynes and sulfoxides.

A variety of functionalized sulfonium ylides were prepared in good yields through MnSO4-promoted S-O bond cleavage from activated alkynes and sulfoxides. Experimental results showed that the MnSO4 catalyst played important roles in accelerating the reaction and promoting the [1,3]-rearrangement of the S-O bond. Furthermore, the product was easily obtained on a gram scale by simple recrystallization without column chromatography. The obtained product can be converted to new sulfonium ylides and undergo cycloaddition with an alkyne to afford a trisubstituted furan scaffold.

Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii.

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles.IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

Synthesis of N-Aryl Oxindole Nitrones through a Metal-Free Selective N-Arylation Process.

An efficient selective N-arylation of 3-(hydroxyimino)indolin-2-ones with diaryliodonium salts to prepare (Z)-N-aryl oxindole nitrones has been achieved under simple base-mediated conditions. The reaction tolerated a variety of diaryliodonium salts with diverse and sensitive functional groups. Studies on the oxime structures revealed that the pyrroline ring and carbonyl group in 3-(hydroxyimino)indolin-2-ones played important roles in the selective N-arylation process. The N-aryl oxindole nitrones could be prepared rapidly and easily at the gram scale.

Copper-Catalyzed Selective N-Vinylation of 3-(Hydroxyimino)indolin-2-ones with Alkenyl Boronic Acids: Synthesis of N-Vinyl Nitrones and Spirooxindoles.

A copper-catalyzed selective cross-coupling reaction of 3-(hydroxyimino)indolin-2-ones with alkenyl boronic acids to access (E)-N-vinyl oxindole nitrones has been achieved under mild conditions. The studies showed that catalytic copper salt selectively gave mono N-vinylation products, while 2.0 equiv of copper salt provided double N-vinylation products. The control experiments revealed that the carbonyl group in 3-(hydroxyimino)indolin-2-one played important roles on N-vinylation. Furthermore, the prepared N-vinyl oxindole nitrones could be converted to spirooxindoles in good yields under thermal conditions.

Cycloaddition of Fluorenone N-Aryl Nitrones with Methylenecyclopropanes and Sequential 1,3-Rearrangement: An Entry to Synthesis of Spirofluorenylpiperidin-4-ones.

A facile synthesis of various spirofluorenylpiperidin-4-ones has been achieved in good yields from fluorenone N-aryl nitrones and methylenecyclopropanes. This method involved an initial cycloaddition to form a 5-spirocyclopropane-isoxazoline, which underwent a highly selective 1,3-rearrangement to give the desired product. The stereochemistry of the spirofluorenylpiperidin-4-one could be controlled by the cycloaddition and sequential rearrangement strategy. Furthermore, the spirofluorenylpiperidin-4-ones could be not only prepared in one-pot procedure but also converted to useful scaffolds by reduction or oxidation conditions.

Novel Flavones from the Root of Phytolacca acinosa Roxb.

Two new flavones, 6,7-methylenedioxy-4-hydroxypeltogynan-7'-one (1), cochliophilin B (2), as well as two known ones, cochliophilin A (3) and 6-methoxy-7-hydroxy flavone (4), were isolated from the ethanol extract of the root of Phytolacca acinosa Roxb. Compound 1 is a flavanol framework with one δ-lactone unit, which is rather rare in nature. The structures of the new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D- and 2D-NMR) analyses, the absolute configuration of 1 was established by comparing experimental and calculated electronic circular dichroism spectra. The structures of known compounds were fixed by comparison with literatures data. Compounds 2 and 4 showed modest inhibitory activities against BEL-7402 cell line, with IC50 values of 28.22 and 39.16 µmol/L, respectively.

Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.

The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (ΔpyrF). A markerless gene disruption vector was constructed by incorporating a pyrF-sim (R) cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon-nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii.

Tandem C-O and C-N Bonds Formation Through O-Arylation and [3,3]-Rearrangement by Diaryliodonium Salts: Synthesis of N-Aryl Benzo[1,2,3]triazin-4(1H)-one Derivatives.

Metal-free O-arylation and [3, 3]-rearrangement have been shown as an efficient strategy to construct new C-O and C-N bonds in one-pot reactions. The method was used to prepare N-aryl benzo[1,2,3]triazin-4(1H)-one derivatives in good yields from N-hydroxy benzo[1,2,3]triazin-4(3H)-one and diaryliodonium salts. The reaction was tolerated a variety of sensitive functional groups such as iodine, nitro, ester, and aldehyde groups. A rational mechanism was proposed based on the experimental results, and the reaction was easily up to gram scale.

(Diacetoxyiodo)benzene-Mediated Reaction of Ethynylcarbinols: Entry to α,α'-Diacetoxy Ketones and Glycerol Derivatives.

Efficient access to α,α'-diacetoxy ketones has been developed from ethynylcarbinols and PhI(OAc)2. A plausible mechanism for this was proposed on the basis of experimental studies. The usefulness of α,α'-diacetoxy ketone products has been documented, and glycerol derivatives can be easily synthesized in good yields via a one-pot reaction.

Synthesis of α,ß-Unsaturated N-Aryl Ketonitrones from Oximes and Diaryliodonium Salts: Observation of a Metal-Free N-Arylation Process.

An efficient transition-metal-free method for the preparation of α,ß-unsaturated N-aryl ketonitrones under mild conditions has been developed. This reaction shows good functional group tolerance for both electron-rich and electron-deficient substituents on both oximes and diaryliodonium salts. Two examples of gram-scale preparations have been realized in good yields. Further transformations of these nitrones to different N-heterocycles have been demonstrated. DFT calculations suggest that N-arylation products are formed by [1,3]-phenyl migration of an O-coordinated oximate complex via a four-centered transition state, while the O-arylation products are formed by [1,3]-phenyl migration of a N-coordinated oximate complex.

Copper(I)-Catalyzed Dearomatization of Benzofurans with 2-(Chloromethyl)anilines through Radical Addition and Cyclization Cascade.

Herein, we described a copper(I)-catalyzed dearomatization of benzofurans with 2-(chloromethyl)anilines to prepare various tetrahydrobenzofuro[3,2-b]quinolines and 2-(quinolin-2-yl)phenols in good to excellent yields through radical addition and an intramolecular cyclization process. Mechanistic studies revealed that 2-(chloromethyl)anilines served as radical precursors. The present method features broad substrate scope, good functional group tolerance, quinoline scaffold diversity, and radical addition dearomatization of benzofurans.