Development of small-molecular-based radiotracers for PET imaging of PD-L1 expression and guiding the PD-L1 therapeutics.

PURPOSE: Programmed cell death protein ligand 1 (PD-L1) is a crucial biomarker for immunotherapy. However, nearly 70% of patients do not respond to PD-L1 immune checkpoint therapy. Accurate monitoring of PD-L1 expression and quantification of target binding during treatment are essential. In this study, a series of small-molecule radiotracers were developed to assess PD-L1 expression and direct immunotherapy. METHODS: Radiotracers of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were designed based on a 2-methyl-3-biphenyl methanol scaffold and successfully synthesized. Cellular experiments and molecular docking assays were performed to determine their specificity for PD-L1. PD-L1 status was investigated via positron emission tomography (PET) imaging in MC38 tumor models. PET imaging of [68Ga]Ga-D-pep-PMED was performed to noninvasively quantify PD-L1 blocking using an anti-mouse PD-L1 antibody (PD-L1 mAb). RESULTS: The radiosyntheses of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were achieved with radiochemical yields of 87 ± 6%, 82 ± 4%, and 79 ± 9%, respectively. In vitro competition assays demonstrated their high affinities (the IC50 values of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were 90.66 ± 1.24, 160.8 ± 1.35, and 51.6 ± 1.32 nM, respectively). At 120 min postinjection (p.i.) of the radiotracers, MC38 tumors displayed optimized tumor-to-muscle ratios for all radioligands. Owing to its hydrophilic modification, [68Ga]Ga-D-pep-PMED had the highest target-to-nontarget (T/NT) ratio of approximately 6.2 ± 1.2. Interestingly, the tumor/liver ratio was hardly affected by different concentrations of the inhibitor BMS202. We then evaluated the impacts of dose and time on accessible PD-L1 levels in the tumor during anti-mouse PD-L1 antibody treatment. The tumor uptake of [68Ga]Ga-D-pep-PMED significantly decreased with increasing PD-L1 mAb dose. Moreover, after 8 days of treatment with a single antibody, the uptake of [68Ga]Ga-D-pep-PMED in the tumor significantly increased but remained lower than that in the saline group. CONCLUSION: PET imaging with [68Ga]Ga-D-pep-PMED, a small-molecule radiotracer, is a promising tool for evaluating PD-L1 expression and quantifying the target blockade of PD-L1 to assist in the development of effective therapeutic regimens.

Synthesis, preclinical, and initial clinical evaluation of integrin αVß3 and gastrin-releasing peptide receptor (GRPR) dual-targeting radiotracer [68Ga]Ga-RGD-RM26-03.

Integrin receptor αvß3 and gastrin-releasing peptide receptor (GRPR) expression of tumors could be detected using PET imaging with radiolabeled Arg-Gly-Asp (RGD) and the antagonistic bombesin analog RM26, respectively. The purpose of this study was to investigate the dual receptor-targeting property of the heterodimer RGD-RM26-03 (denoted as LNC1015), demonstrate the tumor diagnostic value of [68Ga]Ga-LNC1015 in preclinical experiments, and evaluate its preliminary clinical feasibility. METHODS: LNC1015 was designed and synthesized by linking cyclic RGD and the RM26 peptide. Preclinical pharmacokinetics were detected in a PC3 xenograft model using microPET and biodistribution studies. The clinical feasibility of [68Ga]Ga-LNC1015 PET/CT was performed in patients with breast cancer, and the results were compared with those of 18F-fluorodeoxyglucose (FDG). RESULTS: [68Ga]Ga-LNC1015 had good stability in saline for at least 2 h, and favorable binding affinity and specificity were demonstrated in vitro and in vivo. The tumor uptake and retention of [68Ga]Ga-LNC1015 during PET imaging were improved compared with its monomeric counterparts [68Ga]Ga-RGD and [68Ga]Ga-RM26 at all the time points examined. In our initial clinical studies, the tumor uptake and tumor-to-background ratio (TBR) of primary and metastatic lesions in [68Ga]Ga-LNC1015 PET/CT were significantly higher than those in [18F]FDG PET/CT, resulting in high lesion detection rate and tumor delineation. CONCLUSION: The dual targeting radiotracer [68Ga]Ga-LNC1015 showed significantly improved tumor uptake and retention, as well as lower liver uptake than [68Ga]Ga-RGD and [68Ga]Ga-RM26 monomer. The first-in-human study showed high TBRs in patients, suggesting favorable pharmacokinetics and high clinical feasibility for PET/CT imaging of cancer.

18F-Labeled Amidobenzimidazole Analogue for Visualizing STING Expression in Tumor.

The stimulator of interferon genes (STING) is pivotal in mediating STING-dependent type I interferon production, which is crucial for enhancing tumor rejection. Visualizing STING within the tumor microenvironment is valuable for STING-related treatments, yet the availability of suitable STING imaging probes is limited. In this study, we developed [18F]AlF-ABI, a novel 18F-labeled agent featuring an amidobenzimidazole core structure, for positron emission tomography (PET) imaging of STING in B16F10 and CT26 tumors. [18F]AlF-ABI was synthesized with a decay-corrected radiochemical yield of 38.0 ± 7.9% and radiochemical purity exceeding 97%. The probe exhibited a nanomolar STING binding affinity (KD = 35.6 nM). Upon administration, [18F]AlF-ABI rapidly accumulated at tumor sites, demonstrating significantly higher uptake in B16F10 tumors compared to CT26 tumors, consistent with STING immunofluorescence patterns. Specificity was further validated through in vitro cell experiments and in vivo blocking PET imaging. These findings suggest that [18F]AlF-ABI holds promise as an effective agent for visualizing STING in the tumor microenvironment.

A proof-of-concept study on bioorthogonal-based pretargeting and signal amplify radiotheranostic strategy.

BACKGROUND: Radiotheranostics differs from the vast majority of other cancer therapies in its capacity for simultaneous imaging and therapy, and it is becoming more widely implemented. A balance between diagnostic and treatment requirements is essential for achieving effective radiotheranostics. Herein, we propose a proof-of-concept strategy aiming to address the profound differences in the specific requirements of the diagnosis and treatment of radiotheranostics. RESULTS: To validate the concept, we designed an s-tetrazine (Tz) conjugated prostate-specific membrane antigen (PSMA) ligand (DOTA-PSMA-Tz) for 68Ga or 177Lu radiolabeling and tumor radiotheranostics, a trans-cyclooctene (TCO) modified Pd@Au nanoplates (Pd@Au-PEG-TCO) for signal amplification, respectively. We then demonstrated this radiotheranostic strategy in the tumor-bearing mice with the following three-step procedures: (1) i.v. injection of the [68Ga]Ga-PSMA-Tz for diagnosis; (2) i.v. injection of the signal amplification module Pd@Au-PEG-TCO; (3) i.v. injection of the [177Lu]Lu-PSMA-Tz for therapy. Firstly, this strategy was demonstrated in 22Rv1 tumor-bearing mice via positron emission tomography (PET) imaging with [68Ga]Ga-PSMA-Tz. We observed significantly higher tumor uptake (11.5 ± 0.8%ID/g) with the injection of Pd@Au-PEG-TCO than with the injection [68Ga]Ga-PSMA-Tz alone (5.5 ± 0.9%ID/g). Furthermore, we validated this strategy through biodistribution studies of [177Lu]Lu-PSMA-Tz, with the injection of the signal amplification module, approximately five-fold higher tumor uptake of [177Lu]Lu-PSMA-Tz (24.33 ± 2.53% ID/g) was obtained when compared to [177Lu]Lu-PSMA-Tz alone (5.19 ± 0.26%ID/g) at 48 h post-injection. CONCLUSION: In summary, the proposed strategy has the potential to expand the toolbox of pretargeted radiotherapy in the field of theranostics.

Progressive Optimization of Lanthanide Nanoparticle Scintillators for Enhanced Triple-Activated Radioluminescence Imaging.

Lanthanide nanoparticle (LnNP) scintillators exhibit huge potential in achieving radionuclide-activated luminescence (radioluminescence, RL). However, their structure-activity relationship remains largely unexplored. Herein, progressive optimization of LnNP scintillators is presented to unveil their structure-dependent RL property and enhance their RL output efficiency. Benefiting from the favorable host matrix and the luminescence-protective effect of core-shell engineering, NaGdF4 : 15 %Eu@NaLuF4 nanoparticle scintillators with tailored structures emerged as the top candidates. Living imaging experiments based on optimal LnNP scintillators validated the feasibility of laser-free continuous RL activated by clinical radiopharmaceuticals for tumor multiplex visualization. This research provides unprecedented insights into the rational design of LnNP scintillators, which would enable efficient energy conversion from Cerenkov luminescence, γ-radiation, and ß-electrons into visible photon signals, thus establishing a robust nanotechnology-aided approach for tumor-directed radio-phototheranostics.

In Vivo Comparative Study of Radioiodinated Folate Receptor Targeting Albumin Probes for Atherosclerosis Plaque Imaging.

The objective of this study is to compare a series of albumin-based folate radiotracers for the potential imaging of folate receptor (FR) positive macrophages in advanced atherosclerotic plaques. Diversified radioiodinated FR-targeting albumin-binding probes ([131I]IBAbHF, [131I]IBNHF, and [131I]HF) were developed through various strategies. Among the three radiotracers, [131I]IBAbHF and [131I]IBNHF showed excellent in vitro stability (>98%) in saline and PBS 7.4 for 24 h. Also, good stability of [131I]IBNHF in mouse serum albumin was monitored using an HSA ELISA kit. The experiments in Raw264.7 macrophages activated by ox-LDL confirmed the specificity of tracers for FR-ß. Biodistribution studies of radiotracers were performed to verify the prolonged blood half-life. Prolonged blood half-lives of [131I]IBAbHF, [131I]HF, and [131I]IBNHF were 17.26 ± 4.29, 6.33 ± 2.64, and 5.50 ± 1.26 h, respectively. SPECT-CT imaging of ApoE-/- mice at different stages was performed to evaluate the progression and monitor the prognosis of AS. Evident [131I]IBNHF uptake in atherosclerotic lesions could be observed along with a low background signal. In summary, we demonstrated a proof-of-concept of albumin-based radioligands for FR-targeting atherosclerosis imaging and found that different incorporation of radioiodinated groups resulted in different pharmacokinetic properties. Among these candidate compounds, [131I]IBNHF would be a satisfactory radiotracer for SPECT imaging of atherosclerosis.

Development of [177Lu]Lu-LNC1003 for radioligand therapy of prostate cancer with a moderate level of PSMA expression.

PURPOSE: Evans blue as an albumin binder has been widely used to improve pharmacokinetics and enhance tumor uptake of radioligands, including prostate-specific membrane antigen (PSMA) targeting agents. The goal of this study is to develop an optimal Evans blue-modified radiotherapeutic agent that could maximize the absolute tumor uptake and tumor absorbed dose thus the therapeutic efficacy to allow treatment of tumors even with moderate level of PSMA expression. METHODS: [177Lu]Lu-LNC1003 was synthesized based on PSMA-targeting agent and Evans blue. Binding affinity and PSMA targeting specificity were verified through cell uptake and competition binding assay in 22Rv1 tumor model that has moderate level of PSMA expression. SPECT/CT imaging and biodistribution studies in 22Rv1 tumor-bearing mice were performed to evaluate the preclinical pharmacokinetics. Radioligand therapy studies were conducted to systematically assess the therapeutic effect of [177Lu]Lu-LNC1003. RESULTS: LNC1003 showed high binding affinity (IC50 = 10.77 nM) to PSMA in vitro, which was comparable with that of PSMA-617 (IC50 = 27.49 nM) and EB-PSMA-617 (IC50 = 7.91 nM). SPECT imaging of [177Lu]Lu-LNC1003 demonstrated significantly improved tumor uptake and retention as compared with [177Lu]Lu-EB-PSMA and [177Lu]Lu-PSMA-617, making it suitable for prostate cancer therapy. Biodistribution studies further confirmed the remarkably higher tumor uptake of [177Lu]Lu-LNC1003 (138.87 ± 26.53%ID/g) over [177Lu]Lu-EB-PSMA-617 (29.89 ± 8.86%ID/g) and [177Lu]Lu-PSMA-617 (4.28 ± 0.25%ID/g) at 24 h post-injection. Targeted radioligand therapy results showed noteworthy inhibition of 22Rv1 tumor growth after administration of a single dose of 18.5 MBq [177Lu]Lu-LNC1003. There was no obvious antitumor effect after [177Lu]Lu-PSMA-617 treatment under the same condition. CONCLUSION: In this study, [177Lu]Lu-LNC1003 was successfully synthesized with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With greatly enhanced tumor uptake and retention, [177Lu]Lu-LNC1003 has the potential to improve therapeutic efficacy using significantly lower dosages and less cycles of 177Lu that promises clinical translation to treat prostate cancer with various levels of PSMA expression.

Synthesis and Preclinical Evaluation of a 68Ga-Labeled Pyridine-Based Benzamide Dimer for Malignant Melanoma Imaging.

Benzamide (BZA), a small molecule that can freely cross cell membranes and bind to melanin, has served as an effective targeting group for melanoma theranostics. In this study, a novel pyridine-based BZA dimer (denoted as H-2) was labeled with 68Ga ([68Ga]Ga-H-2) for positron emission tomography (PET) imaging of malignant melanomas. [68Ga]Ga-H-2 was obtained with high radiochemical yield (98.0 ± 2.0%) and satisfactory radiochemical purity (>95.0%). The specificity and affinity of [68Ga]Ga-H-2 were confirmed in melanoma B16F10 cells and in vivo PET imaging of multiple tumor models (B16F10 tumors, A375 melanoma, and lung metastases). Monomeric [68Ga]Ga-H-1 was prepared as a control radiotracer to verify the effects of the molecular structure on pharmacokinetics. The values of the lipid-water partition coefficient of [68Ga]Ga-H-2 and [68Ga]Ga-H-1 demonstrated hydrophilicity with log P = -2.37 ± 0.07 and -2.02 ± 0.09, respectively. PET imaging and biodistribution showed a higher uptake of [68Ga]Ga-H-2 in B16F10 primary and metastatic melanomas than that in A375 melanomas. However, the relatively low uptake of monomeric [68Ga]Ga-H-1 in B16F10 tumors and high accumulation in nontarget organs resulted in poor PET imaging quality. This study demonstrates the synthesis and preclinical evaluation of the novel pyridine-based BZA dimer [68Ga]Ga-H-2 and indicates that the dimer tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma.

Longitudinal MicroSPECT Imaging of Systemic Sclerosis Model Mice with [99mTc]Tc-HYNFA via Folate Receptor Targeting.

Noninvasive single-photon emission computed tomography (SPECT) imaging with [99mTc]Tc-HYNFA via folate receptor (FR) targeting was proposed to assess the inflammation and therapeutic effect of systemic sclerosis (SSc) in model mice. The radiochemical yield and purity of [99mTc]Tc-HYNFA were over 95%, with a specific activity of about 9.36 ± 0.17 MBq/nmol. At the end of induction, the uptake ratios of bleomycin-injected regions on the back-to-muscle (R/M) and lung-to-muscle (L/M) derived from SPECT images were 7.27 ± 0.50 and 4.25 ± 0.15, respectively. The radioactivity uptakes could be blocked by excessive folic acid (FA), and R/M and L/M obviously decreased to 2.78 ± 0.57 and 2.51 ± 0.79, respectively. R/M (2.22 ± 0.71) and L/M (1.62 ± 0.28) decreased very close to those of the control mice group (R/M = 1.99 ± 0.36, L/M = 1.50 ± 0.14) when macrophages had been depleted in advance. After being treated with cyclophosphamide (CTX) or methotrexate (MTX), R/M and L/M decreased to 3.58 ± 0.52 and 2.03 ± 0.32 (CTX treatment) or 2.48 ± 0.64 and 1.83 ± 0.06 (MTX treatment). R/M and L/M were highly correlated with pathological changes. The trend of hydroxyproline content in lungs at the later non-inflammatory phase of each group was similar to the uptake values of the lung in the 4th week from the beginning of induction. [99mTc]Tc-HYNFA had an ideal uptake in SSc lesions. R/M and L/M had a high consistency with pathological changes. SPECT imaging-targeted FR could monitor the therapeutic effect of CTX and MTX. It is expected to be an effective means to evaluate SSc.

Development of 18F-Labeled Acridone Analogue for Tumor Imaging via Stimulator of Interferon Genes Targeting.

The stimulator of interferon genes (STING) is a pivotal protein in the production of STING-dependent type I interferon, which has the potential to enhance tumor rejection. The visualization of STING in the tumor microenvironment is valuable for STING-related treatments, but few STING imaging probes have been reported to date. In this study, we developed a novel 18F-labeled agent ([18F]F-CRI1) with an acridone core structure for the positron emission tomography (PET) imaging of STING in CT26 tumors. The probe was successfully prepared with a nanomolar STING binding affinity of Kd = 40.62 nM. [18F]F-CRI1 accumulated quickly in the tumor sites and its uptake reached a maximum of 3.02 ± 0.42% ID/g after 1 h i.v. injection. The specificity of [18F]F-CRI1 was confirmed both in in vitro cell uptake and in vivo PET imaging by blocking studies. Our findings suggest that [18F]F-CRI1 may be a potential agent for visualizing STING in the tumor microenvironment.

Synergism of 64Cu-Labeled RGD with Anti-PD-L1 Immunotherapy for the Long-Acting Antitumor Effect.

We put forward a novel targeting-triggering-therapy (TTT) scheme that combines 64Cu-based targeted radionuclide therapy (TRT) with programmed death-ligand 1 (PD-L1)-based immunotherapy for enhancing therapeutic efficacy. The αvß3 integrin-targeted 64Cu-DOTA-EB-cRGDfK (64Cu-DER) was synthesized. Flow cytometry, immunofluorescence staining, and RT-qPCR were performed to verify PD-L1 upregulation after irradiation with 64Cu-DER. Positron emission tomography imaging was performed to investigate the prominent tumor retention property of 64Cu-DER. In the MC38 tumor model, anti-PD-L1 antibody (αPD-L1 mAb) was delivered in a concurrent or sequential manner after 64Cu-DER was injected, followed by the testing of changes in tumor microenvironment (TME). PD-L1 was upregulated in a time- and dose-dependent manner after being induced by 64Cu-DER. The combination of 64Cu-DER TRT (925 MBq/kg) and αPD-L1 mAb (10 mg/kg) resulted in significant delay in tumor growth and protected against tumor rechallenge. Blockade of PD-L1 at 4 h after 64Cu-DER TRT (64Cu-DER + αPD-L1 mAb @ 4 h combination group) was able to achieve 100% survival rate, prevent tumor relapse, and evidently prolong the survival of mice. In summary, the combination of 64Cu-DER and αPD-L1 mAb in a time-dependent manner could be a promising approach to improve therapeutic efficacy. Understandably, this strategy has the potential to extend the scope of 64Cu-based TTT and merits translation into clinical practice for the better management of immune checkpoint blockade immunotherapy.

MicroPET imaging of bacterial infection with nitroreductase-specific responsive 18F-labelled nitrogen mustard analogues.

PURPOSE: Bacterial infection and antibiotic resistance are serious threats to human health. This study aimed to develop two novel radiotracers, 18F-NTRP and 18F-NCRP, that possess a specific nitroreductase (NTR) response to image deep-seated bacterial infections using positron emission tomography (PET). This method can distinguish infection from sterile inflammation. METHODS: 18F-NTRP and 18F-NCRP were synthesized via a one-step method; all the steps usually involved in tracer radiosynthesis were successfully adapted in the All-In-One automated module. After the physiochemical properties of 18F-NTRP and 18F-NCRP were characterized, their specificity and selectivity for NTR were verified in E. coli and S. aureus. The ex vivo biodistribution of the tracers was evaluated in normal mice. MicroPET-CT imaging was performed in mouse models of bacterial infection and inflammation after the administration of 18F-NTRP or 18F-NCRP. RESULTS: Fully automated radiosynthesis of 18F-NTRP and 18F-NCRP was achieved within 90-110 min with overall decay-uncorrected, isolated radiochemical yields of 21.24 ± 4.25% and 11.3 ± 3.78%, respectively. The molar activities of 18F-NTRP and 18F-NCRP were 320 ± 40 GBq/µmol and 275 ± 33 GBq/µmol, respectively. In addition, 18F-NTRP and 18F-NCRP exhibited high selectivity and specificity for NTR response. PET-CT imaging in bacteria-infected mouse models with 18F-NTRP or 18F-NCRP showed significant radioactivity uptake in either E. coli- or S. aureus-infected muscles. The uptake for E. coli-infected muscles, 2.4 ± 0.2%ID/g with 18F-NTRP and 4.05 ± 0.49%ID/g with 18F-NCRP, was up to three times greater than that for uninfected control muscles. Furthermore, for both 18F-NTRP and 18F-NCRP, the uptake in bacterial infection was 2.6 times higher than that in sterile inflammation, allowing an effective distinction of infection from inflammation. CONCLUSION: 18F-NTRP and 18F-NCRP are worth further investigation to verify their potential clinical application for distinguishing bacterial infection from sterile inflammation via their specific NTR responsiveness.

A radioiodinated FR-ß-targeted tracer with improved pharmacokinetics through modification with an albumin binder for imaging of macrophages in AS and NAFL.

PURPOSE: The formation of advanced plaques, which is characterized by the uninterrupted aggregation of macrophages with high expression of folate receptor-ß (FR-ß), is observed in several concomitant metabolic syndromes. The objective of this study was to develop a novel FR-ß-targeted single-photon emission computed tomography (SPECT) radiotracer and validate its application to the noninvasive detection of atherosclerosis (AS) plaque and non-alcoholic fatty liver (NAFL). METHODS: Two radioiodinated probes, [131I]IPBF and [131I]IBF, were developed, and cell uptake studies were used to identify their specific targets for activated macrophages. Biodistribution in normal mice was performed to obtain the pharmacokinetic information of the probes. Apolipoprotein E knockout (ApoE-/-) mice with atherosclerotic aortas were induced by a high-fat and high-cholesterol (HFHC) diet. To investigate the affinity of radiotracers to FR-ß, Kd values were determined using in vitro assays. In addition, the assessments of the aorta in the ApoE-/- mice at different stages were performed using in vivo SPECT/CT imaging, and the findings were compared by histology. RESULTS: Both [131I]IPBF and [131I]IBF were synthesized with > 95% radiochemical purity and up to 3 MBq/nmol molar activity. In vitro assay of [131I]IPBF showed a moderate binding affinity to plasma proteins and specific uptake in activated macrophages. The prolonged blood elimination half-life (t1/2z) of [131I]IPBF (8.14 h) was observed in a pharmacokinetic study of normal mice, which was significantly longer than that of [131I]IBF (t1/2z = 2.95 h). As expected, the Kd values of [131I]IPBF and [131I]IBF in the Raw 264.7 cells were 43.94 ± 9.83 nM and 61.69 ± 15.19 nM, respectively. SPECT imaging with [131I]IPBF showed a high uptake in advanced plaques and NAFL. Radioactivity in excised aortas examined by ex vivo autoradiography further confirmed the specific uptake of [131I]IPBF in high-risk AS plaques. CONCLUSIONS: In summary, we reported a proof-of-concept study of an albumin-binding folate derivative for macrophage imaging. The FR-ß-targeted probe, [131I]IPBF, significantly prolongs the plasma elimination half-life and has the potential for the monitoring of AS plaques and concomitant fatty liver.

A novel 18F-labeled agonist for PET imaging of stimulator of interferon gene expression in tumor-bearing mice.

PURPOSE: Stimulator of interferon genes (STING) protein plays a vital role in the immune surveillance of tumor microenvironment. Monitoring STING expression in tumors benefits the relevant STING therapy. This study aimed to develop a novel 18F-labeled agonist, dimeric amidobenzimidazole (diABZI), and firstly evaluate the feasibility of noninvasive positron emission tomography (PET) imaging of STING expression in the tumor microenvironment. METHODS: An analog of the STING agonist NOTA-DABI was synthesized and labeled with 18F via Al18F-NOTA complexation (denoted as [18F]F-DABI). Physicochemical properties, STING protein-binding affinity, and specificity of [18F]F-DABI were evaluated using cell uptake and docking assays. In vivo small-animal PET imaging and biodistribution studies of [18F]F-DABI in tumor-bearing mice were performed to verify the pharmacokinetics and tumor targeting ability. The correlation between tumor uptake and STING expression was also analyzed. RESULTS: [18F]F-DABI was produced conveniently with high radiochemical yield (44 ± 15%), radiochemical purity (> 97%) and molar activity (15-30 GBq/µmol). In vitro binding assays demonstrated that [18F]F-DABI has a favorable affinity and specificity for STING with a KD of 12.98 ± 2.07 nM. In vivo studies demonstrated the specificity of [18F]F-DABI for PET imaging of STING expression with B16F10 tumor uptake of 10.93 ± 0.93%ID/g, which was significantly different from that of blocking groups (3.13 ± 0.88%ID/g, ***p < 0.0001). Furthermore, tumor uptake of [18F]F-DABI was well positively correlated with STING expression in different tumor types. Biodistribution results demonstrated that [18F]F-DABI was predominately uptaken in the liver and intestines, indicating its hepatobiliary elimination. CONCLUSION: This proof-of-concept study demonstrated a STING-binding radioligand for PET imaging, which could be used as a potential companion diagnostic tool for related STING-agonist therapies.

Novel Radiolabeled TMTP1 for Long-Acting Hepatocellular Carcinoma Therapeutics.

Currently, the 5-year survival rate for patients with advanced hepatocellular carcinoma (HCC) is very low. Therefore, there is an urgent need to find new strategies for the treatment of HCC. TMTP1 (NVVRQ) is a tumor-homing peptide that has been shown to target a range of highly metastatic tumor cells. In this study, a novel radiotherapeutic probe, [177Lu]Lu-DOTA-EB-TMTP1, was synthesized and used to explore the antitumor efficacy in an HCC tumor model. The albumin-binding TMTP1 radioligand was achieved with >98% radiochemical purity. Long tumor retention property of [177Lu]Lu-DOTA-EB-TMTP1 was exhibited in single photon emission computed tomography (SPECT) imaging and biodistribution study. The [177Lu]Lu-DOTA-EB-TMTP1 showed significant accumulation in the SMMC-7721 HCC tumor with an uptake value of 9.67 ± 1.27 %ID/g at 8 h and a T/M ratio of 6.4. In radiotherapy studies, 30 days after injection of [177Lu]Lu-DOTA-EB-TMTP1, the tumor inhibition rate reached 93.2 ± 0.10 and 94.9 ± 0.04% in the 18.5 and 29.6 MBq high-dose groups, respectively. These preclinical data suggest that [177Lu]Lu-DOTA-EB-TMTP1 may be an effective treatment option for HCC and should be further evaluated in human trials.

Dual Targeting of Integrin αvß3 and Neuropilin-1 Receptors Improves Micropositron Emission Tomography Imaging of Breast Cancer.

The receptors neuropilin-1 (NRP-1) and integrin αvß3 are overexpressed in breast cancer and associated with neovascularization. We synthesized a heterodimeric tracer, 68Ga-DOTA-RGD-ATWLPPR, which simultaneously targets integrin αvß3 and NRP-1 in breast cancer. In this study, we evaluated the diagnostic efficacy of 68Ga-DOTA-RGD-ATWLPPR during micropositron emission tomography (microPET)/X-ray computed tomography (CT) imaging and gamma counting. We evaluated the receptor-binding characteristics and tumor-targeting efficacy of the tracer in vitro and in vivo. Static microPET/CT imaging and gamma counting studies showed that 68Ga-DOTA-RGD-ATWLPPR uptake in MCF-7 tumors is higher than that of monomeric tracers. 68Ga-DOTA-RGD-ATWLPPR uptake could be blocked with excess unlabeled RGD or ATWLPPR, demonstrating the sensitivity and specificity of the tracer. We did not observe bone tracer uptake in vivo, but the data indicated that 68Ga-DOTA-RGD-ATWLPPR is metabolized in the kidneys and the liver uptake is low. In conclusion, 68Ga-DOTA-RGD-ATWLPPR has improved binding affinity, targeting efficiency, and tumor retention time when compared to monomeric tracers, suggesting that it has potential as an imaging probe for breast cancer detection.

PD-L1-Targeted Radionuclide Therapy Combined with αPD-L1 Antibody Immunotherapy Synergistically Improves the Antitumor Effect.

Immune checkpoint blockers (ICBs) targeting programmed death receptor 1 (PD-1) ligand 1 (PD-L1) for immunotherapy have radically reformed oncology. It is of great significance to enhance the response rate of ICB in cancer patients. Here, a radioiodinated anti-PD-L1 antibody (131I-αPD-L1) was developed for PD-L1-targeted single-photon emission computed tomography (SPECT) imaging and αPD-L1 immunotherapy. Flow cytometry and immunofluorescence staining were performed to identify PD-L1 upregulation in a time- and dose-dependent manner after being induced by 131I-αPD-L1. ImmunoSPECT imaging and biodistributions of 131I-αPD-L1 in CT26, MC38, 4T1, and B16F10 tumor models were conducted to visualize the high tumor uptake and low background signal. Compared to monotherapy alone, concurrent administration of αPD-L1 mAb and 131I-αPD-L1 revealed improved tumor control in murine tumor models. The combination of 11.1 MBq of 131I-αPD-L1 and 200 µg of αPD-L1 mAb resulted in significant tumor growth delay and prolonged survival. This radioligand synergized immunotherapy strategy holds great potential for cancer management.

Development of Fibroblast Activation Protein Inhibitor-Based Dimeric Radiotracers with Improved Tumor Retention and Antitumor Efficacy.

Fibroblast activation protein (FAP), a fundamental component of the tumor stroma, is overexpressed in cancer-associated fibroblasts (CAFs). As a promising theranostic probe, we evaluated whether the FAP inhibitor (FAPI) dimer (DOTA-2P[FAPI]2) is more effective than its monomeric analogs for FAP-targeted radionuclide therapy. [68Ga]Ga/[177Lu]Lu-DOTA-2P(FAPI)2 were assayed in a stability study, small-animal positron emission tomography (PET) and single-photon emission computed tomography (SPECT), biodistribution, and radionuclide therapy to comprehensively evaluate their preclinical pharmacokinetics. The pharmacokinetics of [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were determined in FAP-positive hepatocellular carcinoma patient-derived xenografts (PDXs) and HT-1080-FAP cell-derived xenografts (CDXs). [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were stable in phosphate-buffered saline for 4 h. The tumor retention of [68Ga]Ga-DOTA-2P(FAPI)2 was better than that of [68Ga]Ga-FAPI-46 in HT-1080-FAP CDXs, while healthy organs showed low tracer uptake and fast body clearance. In single-photon emission computed tomography, [177Lu]Lu-DOTA-2P(FAPI)2 showed a higher uptake and longer retention for tumors in both PDXs and CDXs from 1-48 h. [177Lu]Lu-DOTA-2P(FAPI)2 showed the best inhibition of tumor growth in PDXs and CDXs. DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared to FAPI-46, which significantly improves the use of FAPI-based vectors for PET imaging and radionuclide therapy. [177Lu]Lu-DOTA-2P(FAPI)2 may be safe and effective for the treatment of FAP-positive malignant tumors.

Radioiodinated 4-(p-Iodophenyl) Butanoic Acid-Modified Estradiol Derivative for ER Targeting SPECT Imaging.

Overexpression of estrogen receptors (ERs) is one of the important characteristics of most breast cancers. We aim to develop a new type of ER-specific radioiodine-labeled estrogen derivative ([131I]IPBA-EE), which was modified with an albumin-specific ligand 4-(p-iodophenyl) butyric acid (IPBA) to improve the metabolic stability and enhance the ER-targeting ability of estrogen. [131I]IPBA-EE can effectively bind to albumin in vitro, and its dissociation constant (Kd = 0.31 µM) is similar to IPBA (Kd = 0.30 µM). The uptake of [131I]IPBA-EE in ER-positive MCF-7 cells (41.81 ± 3.41%) was significantly higher than that in ER-negative MDA-MB-231 cells (8.78 ± 2.37%, ***P < 0.0005) and could be significantly blocked (3.92 ± 0.35%, ***P < 0.0005). The uptakes of [131I]IPBA-EE in rat uterus and ovaries were 5.66 ± 0.34% ID/g and 5.71 ± 2.77% ID/g, respectively, at 1 h p.i., and these uptakes could be blocked by estradiol (uterus: 2.81 ± 0.41% ID/g, *P < 0.05; ovarian: 3.02 ± 0.08% ID/g, *P < 0.05). SPECT/CT imaging showed that ER-positive MCF-7 tumor uptake of [131I]IPBA-EE reached to 6.07 ± 0.20% ID/g at 7 h p.i., which was significantly higher than that of ER-negative MDA-MB-231 tumor (0.87 ± 0.08% ID/g, **P < 0.005) and could be blocked obviously with fulvestrant (1.65 ± 1.56% ID/g, *P < 0.05). In conclusion, a novel radioiodinated estradiol derivative, [131I]IPBA-EE with albumin-binding property and good metabolic stability, was developed to image the ER in breast cancer. This promising ER-targeted probe has the potential to warrant further preclinical investigations.

A Novel Cascade Nanoreactor Integrating Two-Dimensional Pd-Ru Nanozyme, Uricase and Red Blood Cell Membrane for Highly Efficient Hyperuricemia Treatment.

Nanozyme-based cascade reaction has emerged as an effective strategy for disease treatment because of its high efficiency and low side effects. Herein, a new and highly active two-dimensional Pd-Ru nanozyme is prepared and then integrated with uricase and red blood cell (RBC) membrane to fabricate a tandem nanoreactor, Pd-Ru/Uricase@RBC, for hyperuricemia treatment. The designed Pd-Ru/Uricase@RBC nanoreactor displayed not only good stability against extreme pH, temperature and proteolytic degradation, but also long circulation half-life and excellent safety. The nanoreactor can effectively degrade UA by uricase to allantoin and H2 O2 and remove H2 O2 by using Pd-Ru nanosheets (NSs) with the catalase (CAT)-like activity. More importantly, the finally produced O2 from H2 O2 decomposition can in turn facilitate the catalytic oxidation of UA, as the degradation of UA is an O2 consumption process. By integrating the high-efficiency enzymatic activity, long circulation capability, and good biocompatibility, the designed Pd-Ru/Uricase@RBC can effectively and safely treat hyperuricemia without side effects. The study affords a new alternative for the exploration of clinical treatment of hyperuricemia.