68Ga-NC-BCH Whole-Body PET Imaging Rapidly Targets Claudin18.2 in Lesions in Gastrointestinal Cancer Patients.

68Ga-labeled nanobody (68Ga-NC-BCH) is a single-domain antibody-based PET imaging agent. We conducted a first-in-humans study of 68Ga-NC-BCH for PET to determine its in vivo biodistribution, metabolism, radiation dosimetry, safety, and potential for quantifying claudin-18 isoform 2 (CLDN18.2) expression in gastrointestinal cancer patients. Methods: Initially, we synthesized the probe 68Ga-NC-BCH and performed preclinical evaluations on human gastric adenocarcinoma cell lines and xenograft mouse models. Next, we performed a translational study with a pilot cohort of patients with advanced gastrointestinal cancer on a total-body PET/CT scanner. Radiopharmaceutical biodistribution, radiation dosimetry, and the relationship between tumor uptake and CLDN18.2 expression were evaluated. Results: 68Ga-NC-BCH was stably prepared and demonstrated good radiochemical properties. According to preclinical evaluation,68Ga-NC-BCH exhibited rapid blood clearance, high affinity for CLDN18.2, and high specific uptake in CLDN18.2-positive cells and xenograft mouse models. 68Ga-NC-BCH displayed high uptake in the stomach and kidney and slight uptake in the pancreas. Compared with 18F-FDG, 68Ga-NC-BCH showed significant differences in uptake in lesions with different levels of CLDN18.2 expression. Conclusion: A clear correlation was detected between PET SUV and CLDN18.2 expression, suggesting that 68Ga-NC-BCH PET could be used as a companion diagnostic tool for optimizing treatments that target CLDN18.2 in tumors.

Preclinical imaging evaluation of a bispecific antibody targeting hPD1/CTLA4 using humanized mice.

BACKGROUND: The lack of an efficient way to screen patients who are responsive to immunotherapy challenges PD1/CTLA4-targeting cancer treatment. Immunotherapeutic efficacy cannot be clearly determined by peripheral blood analyses, tissue gene markers or CT/MR value. Here, we used a radionuclide and imaging techniques to investigate the novel dual targeted antibody cadonilimab (AK104) in PD1/CTLA4-positive cells in vivo. METHODS: First, humanized PD1/CTLA4 mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to express hPD1/CTLA4 in T-cells. Then, mouse colon cancer MC38-hPD-L1 cell xenografts were established in humanized mice. A bispecific antibody targeting PD1/CTLA4 (AK104) was labeled with radio-nuclide iodine isotopes. Immuno-PET/CT imaging was performed using a bispecific monoclonal antibody (mAb) probe 124I-AK104, developed in-house, to locate PD1+/CTLA4+ tumor-infiltrating T cells and monitor their distribution in mice to evaluate the therapeutic effect. RESULTS: The 124I-AK104 dual-antibody was successfully constructed with ideal radiochemical characteristics, in vitro stability and specificity. The results of immuno-PET showed that 124I-AK104 revealed strong hPD1/CTLA4-positive responses with high specificity in humanized mice. High uptake of 124I-AK104 was observed not only at the tumor site but also in the spleen. Compared with PD1- or CTLA4-targeting mAb imaging, 124I-AK104 imaging had excellent standard uptake values at the tumor site and higher tumor to nontumor (T/NT) ratios. CONCLUSIONS: The results demonstrated the potential of translating 124I-AK104 into a method for screening patients who benefit from immunotherapy and the efficacy, as well as the feasibility, of this method was verified by immuno-PET imaging of humanized mice.

KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis.

Pancreatic ductal adenocarcinoma (PDAC), which has a poor prognosis and nonspecific symptoms and progresses rapidly, is the most common pancreatic cancer type. Inhibitors targeting KRAS G12D and G12C mutations have been pivotal in PDAC treatment. Cancer cells with different KRAS mutations exhibit various degrees of glutamine dependency; in particular, cells with KRAS G12D mutations exhibit increased glutamine uptake. (2S,4R)-4-[18F]FGln has recently been developed for clinical cancer diagnosis and tumor cell metabolism analysis. Thus, we verified the heterogeneity of glutamine dependency in PDAC models with different KRAS mutations by a visual and noninvasive method with (2S,4R)-4-[18F]FGln. Two tumor-bearing mouse models (bearing the KRAS G12D or G12C mutation) were injected with (2S,4R)-4-[18F]FGln, and positron emission tomography (PET) imaging features and biodistribution were observed and analyzed. The SUVmax in the regions of interest (ROI) was significantly higher in PANC-1 (G12D) tumors than in MIA PaCa-2 (G12C) tumors. Biodistribution analysis revealed higher tumor accumulation of (2S,4R)-4-[18F]FGln and other metrics, such as T/M and T/B, in the PANC-1 mouse models compared to those in the MIAPaCa-2 mouse models. In conclusion, PDAC cells with the KRAS G12D and G12C mutations exhibit various degrees of (2S,4R)-4-[18F]FGln uptake, indicating that (2S,4R)-4-[18F]FGln might be applied to detect KRAS G12C and G12D mutations and provide treatment guidance.

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.

An iodine­labelled Antibody-drug conjugate PET probe for noninvasive monitoring of Nectin-4 expression in urothelial carcinoma.

BACKGROUND AND PURPOSE: Some kinds of antibody-drug conjugate (ADC) with high affinity to Nectin-4 have demonstrated breakthrough progress in the third-line setting for bladder cancer. However, many patients are still difficult to benefit from treatment based on the heterogeneity of tumour. As the most advanced auxiliary treatment technology, treatment visualization can most intuitively predict the effectiveness of drug treatment, and timely detect the occurrence of drug resistance. Among them, nuclear medicine molecular probes play an important role in this field. METHODS: 124/125I-EV was prepared by labelling Enfortumad Vedetin (EV), an ADC drugs widely used in clinic targeted Nectin-4, with Na124/125I using N-bromine succinimide as oxidant. The radiochemical purity was analyzed via radio-TLC and bioactivity was measured by enzyme-linked immunosorbent assay. Cell uptake assay and small-animal PET imaging were performed to verified the specificity and targeting. KEY RESULTS: 124/125I-EV was prepared with high labeling yield and radiochemical purity. ELISA assays demonstrated that 124I-EV maintained the same high bioactivity as EV with significantly higher uptake in SW780 cells (Nectin-4 positive, 4.05 ± 0.32 %IA/5 × 105 cells at 8 h) than that in T24 cells (Nectin-4 negative, 1.34 ± 0.18 %IA/5 × 105 cells, p < 0.001). In PET imaging, 124I-EV had a significantly higher accumulation in SW780 tumour than that in T24 tumour and the uptake in SW780 tumour could be specifically blocked when co-injected with cold EV. The signal-to-noise ratio at the tumour site gradually increased with time, and peaked at 72 h. CONCLUSION AND IMPLICATIONS: 124I-EV was successfully prepared with high specificity and binding affinity of Nectin-4. This radioactive probe completely simulates the internal circulation of ADC drugs and tumour uptake and retention, which will greatly improve the clinical application of ADC therapy.

Screening, Construction, and Preliminary Evaluation of CLDN18.2-Specific Peptides for Noninvasive Molecular Imaging.

Recent global clinical trials have shown that CLDN18.2 is an ideal target for the treatment of gastric cancer and that patients with high CLDN18.2 expression can benefit from targeted therapy. Therefore, accurate and comprehensive detection of CLDN18.2 expression is important for patient screening and guidance in anti-CLDN18.2 therapy. Phage display technology was used to screen CLDN18.2-specific peptides from 100 billion libraries. 293TCLDN18.1 cells were used to exclude nonspecific binding and CLDN18.1 binding sequences, while 293TCLDN18.2 cells were used to screen CLDN18.2-specific binding peptides. The monoclonal clones obtained from phage screening were sequenced, and peptides were synthesized based on the sequencing results. Binding specificity and affinity were assessed with a fluorescein isothiocyanate (FITC)-conjugated peptide. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated peptide was also synthesized for 68Ga radiolabeling. The in vitro and in vivo stability, partition coefficients, in vivo molecular imaging, and biodistribution were also characterized. Overall, 54 monoclonal clones were selected after phage display screening. Subsequently, based on the cell ELISA results, CLDN18.2 preference monoclonal clones were selected for deoxyribonucleic acid (DNA) sequencing, and four 7-peptide sequences were obtained after sequence comparison; among them, a peptide named T37 was further validated in vitro and in vivo. The T37 peptide specifically recognized CLDN18.2 but not CLDN18.1 and bound strongly to CLDN18.2-positive cell membranes. The 68Ga-DOTA-T37 probe exhibits good in vitro properties and high stability as a hydrophilic probe; it has high biological safety, and positron emission tomography/computed tomography (PET/CT) studies have shown that it can specifically target CLDN18.2 protein and CLDN18.2-positive tumors in mice. 68Ga-DOTA-T37 demonstrated the superiority and feasibility of using a CLDN18.2-specific probe in PCT/CT imaging, which deserves further development and exploitation.

Construction and preclinical evaluation of a zirconium-89 labelled monoclonal antibody targeting PD-L2 in lung cancer.

OBJECTIVES: The aim of this study was to design a novel tracer targeting programmed cell death-ligand 2 (PD-L2) to dynamically monitor PD-L2 expression and perform preclinical screening to identify patients who may benefit from immune checkpoint inhibitor therapy (ICI) therapy. METHODS: 89Zr labelling of DFO-conjugated PD-L2 antibody (ATL2) was carried out in Na2CO3 buffer at pH 7 (37 °C, 1 h). In vitro stability was analysed using radio-thin layer chromatography (radio-TLC). The affinity of [89Zr]Zr-DFO-ATL2 was evaluated by radio-ELISA. Cell uptake, pharmacokinetic, and biodistribution experiments were used to evaluate the biological properties. Micro-PET/CT imaging with [89Zr]Zr-DFO-ATL2 was conducted at different time points. Immunohistochemical and HE staining studies were carried out using tumour tissues from tumour-bearing mice. RESULTS: The radiochemical yield of [89Zr]Zr-DFO-ATL2 was 65.6 ± 3.9%, and the radiochemical purity (RCP) of the tracer was greater than 99%. The tracer maintained relatively high stability and had a high affinity for the PD-L2 protein (Kd = 31.85 nM, R2 = 0.94). The uptake of [89Zr]Zr-DFO-ATL2 in A549-PD-L2 cells was higher than that in A549 cells at each time point. Micro-PET/CT showed significant uptake in the tumour region of mice bearing tumours derived from A549-PD-L2 (SUVmax = 3.53 ± 0.09 at 96 h) and H2228 (SUVmax = 2.30 ± 0.12 at 48 h) cells. CONCLUSION: The high tumour uptake at early imaging time points demonstrates the feasibility of applying [89Zr]Zr-DFO-ATL2 to image PD-L2 expression in tumours and is encouraging for further clinical application in the screening of patients who may benefit from ICI therapy.

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.

Development of a CLDN18.2-targeting immuno-PET probe for non-invasive imaging in gastrointestinal tumors.

Claudin18.2 (CLDN18.2) is a tight junction protein that is overexpressed in a variety of solid tumors such as gastrointestinal cancer and oesophageal cancer. It has been identified as a promising target and a potential biomarker to diagnose tumor, evaluate efficacy, and determine patient prognosis. TST001 is a recombinant humanized CLDN18.2 antibody that selectively binds to the extracellular loop of human Claudin18.2. In this study, we constructed a solid target radionuclide zirconium-89 (89Zr) labled-TST001 to detect the expression of in the human stomach cancer BGC823CLDN18.2 cell lines. The [89Zr]Zr-desferrioxamine (DFO)-TST001 showed high radiochemical purity (RCP, >99%) and specific activity (24.15 ± 1.34 GBq/µmol), and was stable in 5% human serum albumin, and phosphate buffer saline (>85% RCP at 96 h). The EC50 values of TST001 and DFO-TST001 were as high as 0.413 ± 0.055 and 0.361 ± 0.058 nM (P > 0.05), respectively. The radiotracer had a significantly higher average standard uptake values in CLDN18.2-positive tumors than in CLDN18.2-negative tumors (1.11 ± 0.02 vs. 0.49 ± 0.03, P = 0.0016) 2 days post injection (p.i.). BGC823CLDN18.2 mice models showed high tumor/muscle ratios 96 h p.i. with [89Zr]Zr-DFO-TST001 was much higher than those of the other imaging groups. Immunohistochemistry results showed that BGC823CLDN18.2 tumors were highly positive (+++) for CLDN18.2, while those in the BGC823 group did not express CLDN18.2 (-). The results of ex vivo biodistribution studies showed that there was a higher distribution in the BGC823CLDN18.2 tumor bearing mice (2.05 ± 0.16 %ID/g) than BGC823 mice (0.69 ± 0.02 %ID/g) and blocking group (0.72 ± 0.02 %ID/g). A dosimetry estimation study showed that the effective dose of [89Zr]Zr-DFO-TST001 was 0.0705 mSv/MBq, which is within the range of acceptable doses for nuclear medicine research. Taken together, these results suggest that Good Manufacturing Practices produced by this immuno-positron emission tomography probe can detect CLDN18.2-overexpressing tumors.

One-Minute Iodine Isotope Labeling Technology Enables Noninvasive Tracking and Quantification of Extracellular Vesicles in Tumor Lesions and Intact Animals.

Real-time monitoring of the biological behavior of extracellular vesicles (EVs) in vivo is limited, which hinders its application in biomedicine and clinical translation. A noninvasive imaging strategy could provide us with useful information on EVs' distribution, accumulation and homing in vivo, and pharmacokinetics. In this study, the long half-life radionuclide iodine-124 (124I) was used to directly label umbilical cord mesenchymal stem cell-derived EVs. The resulting probe, namely, 124I-MSC-EVs, was manufactured and ready to use within 1 min. 124I-labeled MSC-EVs had high radiochemical purity (RCP, >99.4%) and stable in 5% human serum album (HSA) with RCP > 95% for 96 h. We demonstrated efficient intracellular internalization of 124I-MSC-EVs in two prostate cancer cell lines (22RV1 and DU145 cell). The uptake rates of 124I-MSC-EVs in human prostate cancer cell lines 22RV1 and DU145 cells were 10.35 ± 0.78 and 2.56 ± 0.21 (AD%) at 4 h. The promising cellular data has prompted us to investigate the biodistribution and in vivo tracking capability of this isotope-based labeling technique in tumor bearing animals. Using positron emission tomography (PET) technology, we showed that the signal from intravenously injected 124I-MSC-EVs mainly accumulated in the heart, liver, spleen, lung, and kidney in healthy kun ming (KM) mice, and the biodistribution study was similar to the imaging results. In the 22RV1 xenograft model, 124I-MSC-EVs accumulated significantly in the tumor after administration, and with the optimal image acquired at 48 h postinjection, the maximum of standard uptake value (SUVmax) of the tumor was 3-fold higher than that of DU145. Taken together, the probe has a high application prospect in immuno-PET imaging of EVs. Our technique provides a powerful and convenient tool for understanding the biological behavior and pharmacokinetic characteristics of EVs in vivo and facilitates the acquirement of comprehensive and objective data for future clinical studies of EVs.

Preclinical Evaluation of a Fibroblast Activation Protein and a Prostate-Specific Membrane Antigen Dual-Targeted Probe for Noninvasive Prostate Cancer Imaging.

Prostate-specific membrane antigen (PSMA) is a prostate cancer target that plays a crucial role in prostate cancer diagnosis and therapy. Herein, a novel dual-targeted imaging probe, [68Ga]Ga-FAPI-PSMA, was prepared by radiolabeling conjugated DOTA-FAPI-PSMA with the short half-life radionuclide gallium-68 (68Ga), which is dedicated to prostate cancer diagnostic imaging. In vitro, [68Ga]Ga-FAPI-PSMA had higher affinity for the PSMA and FAP high-expressing cell lines 22Rv1 and U87 MG with IC50 values of 4.73 and 2.10 nM, respectively, than in the corresponding negative expression cell lines PC3 and A549, and significant differences in cell uptake were also observed. In vivo, [68Ga]Ga-FAPI-PSMA was rapidly cleared from the body, and the estimated radiation dose was relatively low compared with several other FAPI probes. In 22Rv1 and U87 MG tumor xenografts, [68Ga]Ga-FAPI-PSMA rapidly accumulated in tumors after administration, and the best images can be obtained at 1 h postinjection. In conclusion, the dual-targeted probe [68Ga]Ga-FAPI-PSMA was successfully prepared for in vivo prostate cancer PET/CT imaging.

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.

Construction of an Iodine-Labeled CS1001 Antibody for Targeting PD-L1 Detection and Comparison with Low-Molecular-Peptide Micro-PET Imaging.

Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), the research focus in immune checkpoint regulation, play an important role in tumor immunotherapy. Inhibitors of this pathway are also the focus of tumor immunotherapy research. The PD-1/PD-L1 pathway can be blocked by selective binding to PD-L1. Clinical trials have been conducted in a variety of patients with advanced solid tumors. CS1001 is a high-affinity humanized full-length anti-PD-L1 monoclonal antibody with great clinical significance. We constructed a PD-L1-targeted radioactive molecular probe, 124/125I-labeled full-length antibody CS1001, and evaluated its binding specificity and targeting ability to PD-L1 in tumor cells and tumor models. Additionally, a comparison study with 68Ga-WL12, a PD-L1 targeting peptide, was conducted. The binding potency of 125I-CS1001 to human PD-L1 was evaluated by enzyme-linked immunosorbent assay (ELISA), and the Kd value was 52.1 ± 19.3 nM. The cellular uptake of 125I-CS1001 was examined in Chinese hamster ovary cells (CHO) and CHO expressing human PD-L1 (CHO-hPD-L1). At 2 h, the uptake values of 125I-CS1001 in CHO-hPD-L1 without blocking and in the presence of 0.1 mg non-radiolabeled CS1001 were 3.60 ± 0.08 and 0.09 ± 0.005 (%AD/2 × 105 cells, p < 0.001). Micro-PET imaging was performed between 8 to 192 h after injection of 124I-CS1001 into normal KM mice and CHO-hPD-L1 and HeLa tumor models. The standard uptake value (SUV) of relevant organs in PET images was calculated by drawing regions of interest (ROI). SUVmean of CHO-hPD-L1 tumors was significantly higher than that of HeLa tumors at 48 h (1.98 ± 0.04 vs 0.73 ± 0.14, p = 0.005). The SUVmean of 124I-CS1001 in CHO-hPD-L1 tumors at 48 h was higher than that of 68Ga-WL12 in CHO-hPD-L1 tumors at 0.5 h (1.98 ± 0.04 vs 1.09 ± 0.1 SUVmean, p = 0.007). In conclusion, this work provides a new method for monitoring and evaluating the in vivo expression of PD-L1 in tumors.

Evaluation of SARS-CoV-2-Neutralizing Nanobody Using Virus Receptor Binding Domain-Administered Model Mice.

Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an urgent requirement for the development of additional diagnostic tools for further analysis of the disease. The isolated nanobody Nb11-59 binds to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) with high affinity to neutralize the virus and block the angiotensin-converting enzyme 2- (ACE2-) RBD interaction. Here, we introduce a novel nanobody-based radiotracer named 68Ga-Nb1159. The radiotracer retained high affinity for the RBD and showed reliable radiochemical characteristics both in vitro and in vivo. Preclinical positron emission tomography (PET) studies of 68Ga-Nb1159 in mice revealed its rapid clearance from circulation and robust uptake into the renal and urinary systems. Fortunately, 68Ga-Nb1159 could specifically reveal the distribution of the RBD in mice. This study also helped to evaluate the pharmacodynamic effects of the neutralizing nanobody. Moreover, 68Ga-Nb1159 may be a promising tool to explore the distribution of the RBD and improve the understanding of the virus. In particular, this study identified a novel molecular radioagent and established a reliable evaluation method for specifically investigating the RBD through noninvasive and visual PET technology.

Construction of a 124I-Labeled Specific Antibody for the Noninvasive Detection of Mesothelin-Overexpressing Tumors.

Mesothelin (MSLN) is a molecular biomarker of many types of solid tumors, such as mesothelioma, pancreatic cancer, and colon cancer. Owing to the significant difference in expression between cancer cells and normal cells, mesothelin has been widely used as a key target in cancer immunotherapy. In this study, we used iodine isotope (nat/124/125I)-labeled mesothelin antibodies to noninvasively detect MSLN expression in mice with LS174T colon cancer. The 124I-labeled MSLN antibody showed a high radiochemical purity (RCP, >99%) and specific activity (20.8-67.8 GBq/µmol) after purification and was stable in 5% HSA and PBS (>95% RCP at 8 days). Western blot analysis indicated that the LS174T cells showed a higher MSLN protein level than the HepG2 cells. The half maximal effective concentration (EC50) values of the MSLN antibody and natI-anti-MSLN were 34.77 ± 3.72 ng/mL and 32.60 ± 2.52 ng/mL (P = 0.63), respectively. The dissociation constant of 124I-anti-MSLN binding to MSLN protein was 16.0 nM. The radiotracer showed a significantly higher uptake in LS174T cells than in HepG2 tumor cells (1.56 ± 0.09 vs 0.81 ± 0.03, P = 0.0016) 2 days postinjection. The LS174T mouse models showed extremely low organ uptake and high tumor uptake 96 h after the injection of 124I-anti-MSLN, and the T/M values were much higher than those of the other imaging groups (10.56 ± 1.20 for 124I-anti-MSLN in LS174T mice vs 3.27 ± 0.20 for 124I-anti-MSLN in HepG2 mice vs 3.53 ± 0.2 for 124I-IgG in LS174T mice). The immunochemical histology results showed that LS174T tumors were strongly positive (+++) for MSLN, while those in the HepG2 group showed slight expression (+). The dosimetry estimation study showed that the effective dose of 124I-anti-MSLN was 0.185 mSv/MBq, which is within the range of acceptable doses for further nuclear medicine translational research. Taken together, these results suggest that this radiotracer has the potential for detecting mesothelin-overexpressing tumors.

Clinical translational evaluation of Al18F-NOTA-FAPI for fibroblast activation protein-targeted tumour imaging.

PURPOSE: In this study, a novel aluminium-[18F]fluoride (Al18F)-labelled 1,4,7­triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated fibroblast activation protein inhibitor (FAPI) probe, named Al18F-NOTA-FAPI, was developed for fibroblast activation protein (FAP)-targeted tumour imaging; it could deliver hundreds of millicuries of radioactivity using automated synthesis. The tumour detection efficacy of Al18F-NOTA-FAPI was further validated in both preclinical and clinical translational studies. METHODS: The radiolabelling procedure of Al18F-NOTA-FAPI was optimized. Cell uptake and competitive binding assays were completed with the U87MG and A549 cell lines to evaluate the affinity and specificity of the Al18F-NOTA-FAPI probe. The biodistribution, pharmacokinetics, radiation dosimetry and tumour imaging efficacy of the Al18F-NOTA-FAPI probe were researched in healthy Kunming (KM) and/or U87MG model mice. After the approval of the ethical committee, the Al18F-NOTA-FAPI probe was translated into the clinic for PET/CT imaging of the first 10 cancer patients. RESULTS: The radiolabelling yield of Al18F-NOTA-FAPI was 33.8 ± 3.2% using manual synthesis (n = 10), with a radiochemical purity over 99% and the specific activity of 9.3-55.5 MBq/nmol. The whole body effective dose of Al18F-NOTA-FAPI was estimated to be 1.24E - 02 mSv/MBq, which was lower than several other FAPI probes (68Ga-FAPI-04, 68Ga-FAPI-46 and 68Ga-FAPI-74). In U87MG tumour-bearing mice, Al18F-NOTA-FAPI showed good tumour detection efficacy based on the results of micro PET/CT imaging and biodistribution studies. In an organ biodistribution study of patients, Al18F-NOTA-FAPI showed a lower SUVmean than 2-[18F]-fluoro-2-deoxy-D-glucose (2-[18F]FDG) in most organs, especially in the liver (1.1 ± 0.2 vs. 2.0 ± 0.9), brain (0.1 ± 0.0 vs. 5.9 ± 1.3), and bone marrow (0.9 ± 0.1 vs. 1.7 ± 0.4). Meanwhile, Al18F-NOTA-FAPI did not show extensive bone uptake, and was able to detect more lesions than 2-[18F]FDG in the PET/CT imaging of several patients. CONCLUSION: The Al18F-NOTA-FAPI probe was successfully fabricated and applied in fibroblast activation protein-targeted tumour PET/CT imaging, which showed excellent imaging quality and tumour detection efficacy in U87MG tumour-bearing mice as well as in cancer patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2000038080. Registered 09 September 2020. http://www.chictr.org.cn/showproj.aspx?proj=61192.

Dynamical Coordination of Hand Intrinsic Muscles for Precision Grip in Diabetes Mellitus.

This study investigated the effects of diabetes mellitus (DM) on dynamical coordination of hand intrinsic muscles during precision grip. Precision grip was tested using a custom designed apparatus with stable and unstable loads, during which the surface electromyographic (sEMG) signals of the abductor pollicis brevis (APB) and first dorsal interosseous (FDI) were recorded simultaneously. Recurrence quantification analysis (RQA) was applied to quantify the dynamical structure of sEMG signals of the APB and FDI; and cross recurrence quantification analysis (CRQA) was used to assess the intermuscular coupling between the two intrinsic muscles. This study revealed that the DM altered the dynamical structure of muscle activation for the FDI and the dynamical intermuscular coordination between the APB and FDI during precision grip. A reinforced feedforward mechanism that compensates the loss of sensory feedbacks in DM may be responsible for the stronger intermuscular coupling between the APB and FDI muscles. Sensory deficits in DM remarkably decreased the capacity of online motor adjustment based on sensory feedback, rendering a lower adaptability to the uncertainty of environment. This study shed light on inherent dynamical properties underlying the intrinsic muscle activation and intermuscular coordination for precision grip and the effects of DM on hand sensorimotor function.