A nitroreductase responsive probe for early diagnosis of pulmonary fibrosis disease.

Idiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease. However, the definitive diagnosis of IPF is impeded by the limited capabilities of current diagnostic methods, which may fail to capture the optimal timing for treatment. The main goal of this study is to determine the feasibility of a nitroreductase (NTR) responsive probe, 18F-NCRP, for early detection and deterioration monitoring of IPF. 18F-NCRP was obtained with high radiochemical purity (>95 %). BLM-injured mice were established by intratracheal instillation with bleomycin (BLM) and characterized through histological analysis. Longitudinal PET/CT imaging, biodistribution study and in vitro autoradiography were performed. The correlations between the uptake of 18F-NCRP and mean lung density (tested by CT), as well as histopathological characteristics were analyzed. In PET imaging study, 18F-NCRP exhibited promising efficacy in monitoring the progression of IPF, which was earlier than CT. The ratio of uptake in BLM-injured lung to control lung increased from 1.4-fold on D15 to 2.2-fold on D22. Biodistribution data showed a significant lung uptake of 18F-NCRP in BLM-injured mice. There was a strong positive correlation between the 18F-NCRP uptake in the BLM-injured lungs and the histopathological characteristics. Given that, 18F-NCRP PET imaging of NTR, a promising biomarker for investigating the underlying pathogenic mechanism of IPF, is attainable as well as desirable, which might lay the foundation for establishing an NTR-targeted imaging evaluation system of IPF.

Preclinical Evaluation of 131I/18F-Labeled Covalent Small-Molecule Inhibitors for STING Status Imaging.

The stimulator of interferon genes (STING) is a vital protein to the immune surveillance of the tumor microenvironment. In this study, we develop novel inhibitor-based radioligands and evaluate their feasibility for noninvasive visualization of STING expression in tumor-bearing mice. Analogous compounds to STING inhibitors C170 and C176 were synthesized and labeled with 131I and 18F to attain [131I]I-NFIP and [18F]F-NFEP, respectively. The radiosynthesis was achieved with high radiochemical purity (>95%) and molar activity (28.56-48.89 GBq/µmol). The affinity and specificity of tracers were assessed through cell uptake and docking experiments, demonstrating that [131I]I-NFIP exhibited high specificity for STING, with a cell-based IC50 value of 7.56 nM. Small-animal PET/SPECT imaging and biodistribution studies in tumor-bearing mice models were performed to verify the tracers' pharmacokinetics and tumor-targeting capabilities (n = 3/group). SPECT imaging demonstrated that [131I]I-NFIP rapidly accumulated in the Panc02 tumor quickly at 30 min post-injection, with a tumor-to-muscle (T/M) ratio of 2.03 ± 0.30. This ratio significantly decreased in the blocking group (1.10 ± 0.14, **P < 0.01, n = 3). Furthermore, tumor uptake and the T/M ratio of [131I]I-NFIP were positively associated with STING expression. In summary, [131I]I-NFIP is the first STING-specific inhibitor-based radioligand offering the potential for visualizing STING status in tumors.

Rational Design and Comparison of Novel 99mTc-Labeled FAPI Dimers for Visualization of Multiple Tumor Types.

Recognizing the significance of SPECT in nuclear medicine and the pivotal role of fibroblast activation protein (FAP) in cancer diagnosis and therapy, this study focuses on the development of 99mTc-labeled dimeric HF2 with high tumor uptake and image contrast. The dimeric HF2 was synthesized and radiolabeled with 99mTc in one pot using various coligands (tricine, TPPTS, EDDA, and TPPMS) to yield [99mTc]Tc-TPPTS-HF2, [99mTc]Tc-EDDA-HF2, and [99mTc]Tc-TPPMS-HF2 dimers. SPECT imaging results indicated that [99mTc]Tc-TPPTS-HF2 exhibited higher tumor uptake and tumor-to-normal tissue (T/NT) ratio than [99mTc]Tc-EDDA-HF2 and [99mTc]Tc-TPPMS-HF2. Notably, [99mTc]Tc-TPPTS-HF2 exhibited remarkable tumor accumulation and retention in HT-1080-FAP and U87-MG tumor-bearing mice, thereby surpassing the monomeric [99mTc]Tc-TPPTS-HF. Moreover, [99mTc]Tc-TPPTS-HF2 achieved acceptable T/NT ratios in the hepatocellular carcinoma patient-derived xenograft (HCC-PDX) model, which provided identifiable contrast and imaging quality. In conclusion, this study presents proof-of-concept research on 99mTc-labeled FAP inhibitor dimers for the visualization of multiple tumor types. Among these candidate compounds, [99mTc]Tc-TPPTS-HF2 showed excellent clinical potential, thereby enriching the SPECT tracer toolbox.

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.

68Ga-Labeled TMTP1 Modified with d-Amino Acid for Positron Emission Tomography Diagnosis of Highly Metastatic Hepatocellular Carcinoma.

TMTP1 (NVVRQ) has been proven to selectively target various highly metastatic tumor cells. Nonetheless, existing TMTP1 probes encounter challenges such as rapid blood clearance, limited tumor uptake, and inadequate suitability for therapeutic interventions. To overcome these constraints, we designed and synthesized eight peptide probes, employing innovative chemical modification strategies involving d-amino acid modification and retro-inverso isomerization. Notably, [68Ga]TV2 exhibited particularly impressive performance, displaying an 88.88, 76.90, and 90.32% improvement in uptake at 15, 30, and 60 min, respectively, while maintaining a high target-to-nontarget ratio. Further research has demonstrated that [68Ga]TV2 also exhibits remarkable diagnostic potential for detecting in situ microtumors in the liver. The results suggest that through the implementation of innovative chemical modification strategies, we successfully developed a peptide precursor, NOTA-G-NVvRQ, with specific affinity for highly metastatic tumors, enhanced in vivo pharmacokinetic profile, and heightened stability in vivo, rendering it well suited for prospective investigations in combination therapy studies.

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.

68Ga-Labeled TMTP1 Derivatives with Moderate Hydrophilicity for Positron Emission Tomography of Hepatocellular Carcinoma in High Contrast.

The reported specific positron emission tomography (PET) probes for the diagnosis of highly metastatic hepatocellular carcinoma (HCC) suffer from excessively high background uptake and fast blood clearance. Herein, five 68Ga-labeled polyethylene glycol (PEG)-modified derivatives of the TMTP1 peptide were synthesized. The log D values decreased from -1.70 (non-PEGylated) to -1.97 to -2.94 corresponding to the increase of PEG chain length. Subnanomolar and nanomolar affinities comparable to the non-PEGylated TMTP1 derivative were revealed by the IC50 values in SMMC-7721 cells. [68Ga]Ga-NOTA-PEG2-TMTP1 presented a significantly higher tumor/liver ratio (4.19 ± 0.54, at 30 min post intravenous injection) and tumor/muscle ratio (2.14 ± 0.17) compared to the others and the previously radiolabeled TMTP1 derivatives. Small HCC lesions (<2 mm) in situ were detected with high tumor/liver ratio and low tumor/muscle ratio. The improved pharmacokinetics and blood clearance rate of 68Ga-labeled TMTP1 derivatives indicated that moderate hydrophilicity due to PEGylation contributed to high-contrast PET of HCC.

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.

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.

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.

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.

A Paradigm of Cancer Immunotherapy Based on 2-[18F]FDG and Anti-PD-L1 mAb Combination to Enhance the Antitumor Effect.

PURPOSE: Efforts have been devoted to select eligible candidates for PD-1/PD-L1 immune checkpoint blocker (ICB) immunotherapy. Here, we have a serendipitous finding of positron emission tomography (PET) imaging tracer 2-[18F]FDG as a potential immunomodulator. Therefore, we hypothesize that 2-[18F]FDG could induce PD-L1 expression change and create an immune-favorable microenvironment for tumor immunotherapy. EXPERIMENTAL DESIGN: We designed a series of assays to verify PD-L1 upregulation, and tested immunotherapy regimens based on 2-[18F]FDG and anti-PD-L1 mAb, as monotherapy and in combination, in fully immunocompetent mice of MC38 and CT26 models. PD-L1 expression and tumor microenvironment (TME) changes were analyzed by Western blot, transcriptomics study, and flow-cytometric analysis. RESULTS: PD-L1 was upregulated in a time- and dose-dependent manner after being induced by 2-[18F]FDG. The activation of NF-κB/IRF3 pathway and STAT1/3-IRF1 pathway play crucial parts in modulating PD-L1 expression after DNA damage and repair. Improved αPD-L1 mAb utilization rate and significant tumor growth delay were observed when the personalized therapeutic alliance of 2-[18F]FDG stimulation and ICB was used. In addition, combination of 2-[18F]FDG with αPD-L1 mAb could reprogram a TME from "cold" to "hot," to make low immunoactivity tumors sensitive to ICB therapy. CONCLUSIONS: In summary, this promising paradigm has the potential to expand the traditional tumor theranostics. 2-[18F]FDG-based ICB immunotherapy is highly significant in enhancing antitumor effect. A research of 2-[18F]FDG-based ICB immunotherapy has been proposed to enhance the antitumor effect.

Radioiodinated Ethinylestradiol Derivatives for Estrogen Receptor Targeting Breast Cancer Imaging.

Two novel PEGylated ethinylestradiol (PEG = poly(ethylene glycol)) estrogen receptor (ER) targeting probes [131I]EITE and [131I]MITE were synthesized and evaluated. Both probes had a nanomolar binding affinity to the ER receptor (36.47 nM for [131I]EITE and 61.83 nM for [131I]MITE). They showed high uptake in ER-positive MCF-7 cells and tumors, which could be significantly blocked by a coinjection of excess estradiol. Their ER specificities were further demonstrated by the low uptake in ER-negative MDA-MB-231 cells and tumors. The maximum tumor-to-muscle (T/M) ratios reach to 6.59 for [131I]EITE at 1 h postinjection (p.i.) and to 3.69 for [131I]MITE at 2 h p.i. in MCF-7 tumors. Among these two probes, [131I]EITE showed a faster tumor accumulation and a higher T/M ratio indicating it could be a better candidate for the potential diagnosis of ER-positive breast cancers.

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.

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.

Development and Preclinical Evaluation of Radiolabeled Covalent G12C-Specific Inhibitors for Direct Imaging of the Oncogenic KRAS Mutant.

Although KRAS has been an important target for many cancers, direct inhibition of oncogenic RAS remains challenging. Until recently, covalent KRAS G12C-specific inhibitors have been developed and progressed to the clinics. Nevertheless, not all patients benefit from these covalent inhibitors. At present, identification of candidates for this treatment requires tissue biopsies and gene sequencing, which are invasive, time-consuming, and could be of insufficient quality and limited predictive value owing to tumor heterogeneity. The use of noninvasive molecular imaging techniques such as PET and SPECT for spying KRAS G12C mutation in tumors provide a promising strategy for circumventing these hurdles. In the present study, based on the covalent G12C-specific inhibitor ARS-1620, we sought to develop radiolabeled small molecules for direct imaging of the KRAS mutation status in tumors. [131I]I-ARS-1620 and [18F]F-ARS-1620 were successfully prepared with high radiochemical yield, radiochemical purity, and molar activity. In vitro and in vivo studies have demonstrated the affinity, specificity, and capacity of [131I]I-ARS-1620 for direct imaging of the oncogenic KRAS G12C mutant. This initial attempt allows us to directly screen the KRAS G12C mutant for the first time in vivo.

The development of a Glypican-3-specific binding peptide using in vivo and in vitro two-step phage display screening for the PET imaging of hepatocellular carcinoma.

Glypican-3 (GPC3) is a diagnostic biomarker for hepatocellular carcinoma (HCC). Although numerous designs targeting GPC3 have been reported, the HCC diagnostic agents with specific tumor accumulation and low background, particularly in normal liver tissue, are still in need. Peptides have attracted considerable attention as an imaging probe due to their low immunogenicity, short in vivo circulation time, and acceptable production cost. Herein, a two-step phage display screening approach was performed against GPC3-high expression tumor xenografts in vivo, followed by human recombinant GPC3 protein in vitro. A GPC3-specific binding peptide, named TJ12P2, with the sequence of Ser-Asn-Asp-Arg-Pro-Pro-Asn-Ile-Leu-Gln-Lys-Arg (SNDRPPNILQKR) was identified. The apparent Kd value between TJ12P2 and the GPC3 protein was measured as 158.2 ± 26.25 nM. After 18F labeling, 18F-AlF-NOTA-TJ12P2 was found accumulated in the tumors by positron emission tomography (PET) imaging in two HCC subcutaneous tumor models (HepG2 and SMMC-7721) with high GPC3 expression. Static PET imaging revealed that 18F-AlF-NOTA-TJ12P2 accumulation in the HepG2 and SMMC-7721 tumors reached 1.825 ± 0.296 %ID g-1 and 1.575 ± 0.520 %ID g-1, with tumor-to-muscle ratios of 4.14 ± 0.50 and 4.25 ± 0.25, respectively, at 30 min post-injection (p.i.). Much less accumulation (0.533 ± 0.078 %ID g-1) of the 18F-AlF-NOTA-TJ12P2 was found in the control PC3 tumors with low GPC3 expression. More importantly, no obvious normal liver uptake of TJ12P2 was observed in the abovementioned animal models. As a result, a novel peptide targeting GPC3, TJ12P2, with strong affinity and specificity was identified using a two-step phage display screening technique in the present study. The 18F-AlF-NOTA-TJ12P2 may be a promising PET imaging probe with translational potential for accurate HCC diagnosis.

Syntheses and Preliminary Evaluation of Dual Target PET Probe [18F]-NOTA-Gly3- E (2PEG4-RGD-WH701) for PET Imaging of Breast Cancer.

PURPOSE: Tumor Necrosis Factor Receptor 1 (TNFR1) and integrin αvß3 receptor are overexpressed in breast cancer. We hypothesized that a peptide ligand recognizing both receptors in a single receptor-binding probe would be advantageous. Here, we developed a novel 18F-labeled fusion peptide probe [18F]-NOTA-Gly3- E(2PEG4-RGD-WH701) targeting dual receptors (TNFR1 and αvß3) and evaluated the diagnostic efficacy of this radioactive probe in both MDA-MB-231 and MCF-7 xenograft models in mice. METHODS: The NOTA-conjugated RGD-WH701 analog was radiolabeled with 18F using NOTA-AlF chelation method. We used two PEG4 molecules and Glutamic acid (Glu) to covalently link c(RGDyK) with WH701. Gly3 was also added to further improve the water solubility and pharmacokinetic properties of the probe. The expression of TNFR1 and Integrin αvß3 in MCF-7 and MDA-MB-231 cells was detected by western blot analysis and immunofluorescence staining. The tumor-targeting characteristics of [18F]-NOTA-Gly3-E(2PEG4-RGDWH701) were assessed in nude mice bearing MDA-MB-231 and MCF-7 xenografts. RESULTS: HPLC analysis of the product NOTA-G3-E (2P4-RGD-WH701) revealed a purity >95%. The yield after attenuation correction was approximately 33.5%±2.8% (n=5), and the radiochemical purity was above 95%. The MDA-MB-231 tumor uptake of [18F]-NOTA-Gly3-E(2PEG4-RGD-WH701) was 1.14±0.14%ID/g, as measured by PET at 40min postinjection (p.i.). In comparison, the tumor uptake of [18F]-NOTA-RGD and [18F]- NOTA-WH701 in MDA-MB-231 xenografts was 0.96±0.13%ID/g and 0.93±0.28%ID/g, respectively. The MCF-7 tumor uptake of [18F]-NOTA-Gly3-E(2PEG4-RGD-WH701) was 1.22±0.11%ID/g, as measured by PET at 40min postinjection (p.i.). In comparison, the tumor uptake of [18F]-NOTA-RGD and [18F]-NOTA-WH701 in MCF-7 xenografts was 0.99±0.18%ID/g and 0.57±0.08%ID/g, respectively. CONCLUSION: [18F]AlF-NOTA-Gly3-E(2PEG4-RGD-WH701) was successfully synthesized and labeled with 18F. The results from the microPET/CT and biodistribution studies of [18F]AlF-NOTA-Gly3-E(2PEG4-RGDWH701) showed that the tracer could specifically target TNFR1 and integrin αvß3 receptors.

Radiolabeled Peptides for SPECT and PET Imaging in the Detection of Breast Cancer: Preclinical and Clinical Perspectives.

Breast cancer is the most common cancer in women worldwide. Due to the heterogeneous nature of breast cancer, the optimal treatment and expected response for each patient may not necessarily be universal. Molecular imaging techniques could play an important role in the early detection and targeted therapy evaluation of breast cancer. This review focuses on the development of peptides labeled with SPECT and PET radionuclides for breast cancer imaging. We summarized the current status of radiolabeled peptides for different receptors in breast cancer. The characteristics of radionuclides and major techniques for peptide labeling are also briefly discussed.