18F-Radiolabeling and Evaluation of an AMD3465 Derivative for PET Imaging of CXCR4 in a Mouse Breast Tumor Model.

The exploration of pharmaceutically active agents and positron emission tomography (PET) tracers targeting CXCR4 has been a focal point in cancer research given its pivotal role in the development and progression of various cancers. While significant strides have been made in PET imaging with radiometal-labeled tracers, the landscape of 18F-labeled small molecule tracers remains relatively limited. Herein, we introduce a novel and promising derivative, [18F]SFB-AMD3465, as a targeted PET tracer for CXCR4. The compound was synthesized by modifying the pyridine ring of AMD3465, which was subsequently labeled with 18F using [18F]SFB. The study provides comprehensive insights into the design, synthesis, and biological evaluation of [18F]SFB-AMD3465. In vitro and in vivo assessments demonstrated the CXCR4-dependent, specific, and sensitive uptake of [18F]SFB-AMD3465 in the CXCR4-overexpressing 4T1 cell line and the corresponding xenograft-bearing mouse model. These findings contribute to bridging the gap in 18F-labeled PET tracers for CXCR4 and underscore the potential of [18F]SFB-AMD3465 as a PET radiotracer for in vivo CXCR4 imaging.

Comparison of Al18F- and 68Ga-labeled NOTA-PEG4-LLP2A for PET imaging of very late antigen-4 in melanoma.

Malignant melanoma is an aggressive cancer with poor prognosis. Very late antigen-4 (VLA-4) is overexpressed in melanoma and many other tumors, making it an attractive target for developing molecular diagnostic and therapeutic agents. We compared Al18F- and 68Ga-labeled LLP2A peptides for PET imaging of VLA-4 expression in melanoma. The peptidomimetic ligand LLP2A was modified with chelator 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and the resulting NOTA-PEG4-LLP2A peptide was then radiolabeled with Al18F or 68Ga. The two labeled peptides were assayed for in vitro and in vivo VLA-4 targeting efficiency. Good Al18F and 68Ga radiolabeling yields were achieved, and the resulting PET tracers showed good serum stability. In the in vivo evaluation of the B16F10 xenograft mouse model, both tracers exhibited high accumulation with good contrast in static PET images. Compared with 68Ga-NOTA-PEG4-LLP2A, Al18F-NOTA-PEG4-LLP2A resulted in relatively higher background, including higher liver uptake (1 h: 20.1 ± 2.6 vs. 15.3 ± 1.7%ID/g, P < 0.05; 2 h: 11.0 ± 1.2 vs. 8.0 ± 0.8%ID/g, P < 0.05) and lower tumor-to-blood ratios (2.5 ± 0.4 vs. 3.3 ± 0.5 at 1 h, P < 0.05; 5.1 ± 0.9 vs. 7.3 ± 0.6 at 2 h, P < 0.01) at some time points. The results obtained from the mice blocked with unlabeled peptides and VLA-4-negative A375 xenografts groups confirmed the high specificity of the developed tracers. Despite the relatively high liver uptake, both Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A exhibited high VLA-4 targeting efficacy with comparable in vivo performance, rendering them promising candidates for imaging tumors that overexpress VLA-4.

Evaluation of an Integrin αvß3 and Aminopeptidase N Dual-Receptor Targeting Tracer for Breast Cancer Imaging.

Integrin αvß3 and aminopeptidase N (APN, also known as CD13) are two important targets involved in the regulation of angiogenesis, tumor proliferation, invasion, and metastasis. In this study, we developed a heterodimeric tracer consisting of arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) peptides targeting αvß3 and CD13, respectively, for PET imaging of breast cancer. The NGR peptide was first modified with N3-NOtB2 and then conjugated to BCN-PEG4-c(RGDyK) via copper-free click chemistry. The resulting precursor was purified and radiolabeled with gallium-68. Small-animal PET/CT imaging and post-imaging biodistribution studies were performed in mice bearing human breast cancer MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 xenografts and pulmonary metastases models. The expression levels of αvß3 and CD13 in tumors were checked via immunochemical staining. The heterodimeric tracer was successfully synthesized and radiolabeled with gallium-68 at a molar activity of 45-100 MBq/nmol at the end of synthesis. It demonstrated high in vitro and in vivo stability. In static PET/CT imaging studies, the MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection of 68Ga-NGR-RGD than that of either 68Ga-RGD or 68Ga-NGR alone. High specificity was shown in blocking studies using Arg-Gly-Asp (RGD) and Asp-Gly-Arg (NGR) peptides. The MCF-7 tumor exhibited the highest uptake of 68Ga-NGR-RGD followed by MDA-MB-231, MDA-MB-468, and MX-1 tumors. This was consistent with their expression levels of CD13 and αvß3 as confirmed by western blot and immunohistochemical staining. Metastatic lesions in the lungs were clearly detectable on 68Ga-NGR-RGD PET/CT imaging in mouse models of pulmonary metastases. 68Ga-NGR-RGD, a CD13 and αvß3 dual-receptor targeting tracer, showed higher binding avidities, targeting efficiency, and longer tumor retention time compared with monomeric 68Ga-NGR and 68Ga-RGD. Its promising in vivo performance makes it an ideal candidate for future clinical translation.

Photo-Click-Facilitated Screening Platform for the Development of Hetero-Bivalent Agents with High Potency.

A novel photo-click-based platform has been developed for rapid screening and affinity optimization of heterobivalent agents. This method allows for the efficient selection of high-affinity dual receptor-targeting agents via streamlining tedious organic synthesis and biological evaluation procedures required by traditional approaches. The high-avidity heterobivalent agents targeting both integrin αvß3 and urokinase-type plasminogen activator receptors have been developed using this photo-click-facilitated screening platform. The affinity screening results were further validated by traditional in vitro and in vivo evaluation techniques, reaffirming the reliability of the method. The convenience, rapidity, universality, and robustness of the screening platform, discussed in this report, can greatly facilitate the development of new heterobivalent agents for research and/or clinical applications.

Thermally sensitive fluorescence imaging system for radiofrequency ablation guidance.

Radiofrequency ablation (RFA) has been clinically used as a minimally invasive procedure for the treatment of many solid tumors. However, the current imaging techniques have some shortages in RFA guidance, especially for the assessment of the margin of ablation. Herein, we developed a novel optical imaging platform to guide RFA utilizing fluorescence resonance energy transfer from a thermally sensitive fluorescent protein conjugated to a near-infrared fluorescent dye. Additionally, attaching receptor-targeting ligands further equipped the system with high specificity to tumors overexpressing the targeted receptor.

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

BACKGROUND & AIMS: Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. METHODS: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. RESULTS: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. CONCLUSIONS: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

Synthesis and Evaluation of New Bifunctional Chelators with Phosphonic Acid Arms for Gallium-68 Based PET Imaging in Melanoma.

Due to the increasing use of generator-produced radiometal Gallium-68 (68Ga) in positron-emission tomography/computed tomography (PET/CT), reliable bifunctional chelators that can efficiently incorporate 68Ga3+ into biomolecules are highly desirable. In this study, we synthesized two new bifunctional chelators bearing one or two phosphonic acid functional groups, named p-SCN-PhPr-NE2A1P and p-SCN-PhPr-NE2P1A, with the aim of enabling facile production of 68Ga-based radiopharmaceuticals. Both chelators were successfully conjugated to LLP2A-PEG4, a very late antigen-4 (VLA-4) targeting peptidomimetic ligand, to evaluate their application in 68Ga-based PET imaging. NE2P1A-PEG4-LLP2A exhibited the highest 68Ga3+ binding ability with molar activity of 37 MBq/nmol under mild temperature and neutral pH. Excellent serum stability of 68Ga-NE2P1A-PEG4-LLP2A was observed, which was consistent with the result obtained from density functional theory calculation. The in vitro cell study showed that 68Ga-NE2P1A-PEG4-LLP2A had significantly longer retention in B16F10 cells comparing to the reported retention of 64Cu-NE3TA-PEG4-LLP2A, although the uptake was relatively lower. In the biodistribution and micro-PET/CT imaging studies, high tumor uptake and low background were observed after 68Ga-NE2P1A-PEG4-LLP2A was injected into mice bearing B16F10 tumor xenografts, making it a highly promising radiotracer for noninvasive imaging of VLA-4 receptors overexpressed in melanoma.

Targeted Yttrium 89-Doxorubicin Drug-Eluting Bead-A Safety and Feasibility Pilot Study in a Rabbit Liver Cancer Model.

The purpose of this article is to evaluate feasibility and safety of the cancer targeting (radio)-chemoembolization drug-eluting bead (TRCE-DEB) concept drug SW43-DOX-L-NETA(89Y) DEB for the intra-arterial treatment of VX2 rabbit liver tumors. The treatment compound comprises of the sigma-2 receptor ligand SW43 for cancer targeting, doxorubicin (DOX), and 89yttrium (89Y) as nonradioactive surrogate for therapeutic (yttrium-90, lutetium-177) and imaging (yttrium-86) radioisotopes via the chelator L-NETA. Ten New Zealand white rabbits with VX2 tumor allografts were used. SW43-DOX-89Y was synthesized, loaded onto DEB (100 µL; 100-300 µm), and administered intra-arterially in six rabbits at increasing doses (0.2-1.0 mg/kg). As controls, two rabbits each received either doxorubicin IV (0.3 mg/kg) or no treatment. Consecutive serum analysis for safety and histopathological evaluation after sacrifice were performed. One-Way ANOVA incl. Bonferroni Post-Hoc test was performed to compare groups. Targeted compound synthesis, loading onto DEB, and intra-arterial administration were feasible and successful in all cases. Serum liver enzyme levels increased in a dose dependent manner within 24 h and normalized within 3 days for 0.2/0.6 mg/kg SW43-DOX-89Y loaded onto DEB. The two rabbits treated with 1 mg/kg SW43-DOX-89Y had to be euthanized after 3/24 h due to worsening general condition. Histopathological necrosis increased over time in a dose depended manner with 95-100% tumor necrosis 3-7 days post treatment (0.6 mg/kg). SW43-DOX-89Y loaded onto DEB can be formulated and safely administered at a concentration of 0.6 mg/kg. Loading with radioactive isotopes (e.g., 86yttrium/90yttrium/177lutetium) to synthesize the targeted radio-chemoembolization drug-eluting bead (TRCE-DEB) concept drug is feasible.

Folic Acid-Targeted Etoposide Cubosomes for Theranostic Application of Cancer Cell Imaging and Therapy.

BACKGROUND The aim of this study was to develop a novel Poloxamer-based drug delivery system featuring a tumor-targeting folate moiety, which was expected to provide better targeting properties and therapeutic effects compared with the traditional cubosomes (Cubs). MATERIAL AND METHODS Both folate-modified Cubs containing etoposide (ETP-Cubs-FA) and normal cubic nanoparticles loaded with etoposide (ETP-Cubs) were prepared through the fragmentation of bulk gels under the homogenization condition of 1500 bar, and a mean particle size of around 180 nm was obtained with a narrow size distribution. The cubosomes were further characterized by differential scanning calorimetry (DSC) and Polarized light microscopy (PLM). The release of ETP in vitro from these nanoparticles was found to be 82.5% at 36 h, showing a sustained release property compared with the free drug administration. RESULTS Folate-modified cubosomes exhibited best anti-proliferative activity followed by normal cubosomes and the free drug. A further cell uptake study of Rhodamine B-loaded Cubs-FA (Rh-B-Cubs-FA) showed a marked increase of cellular accumulation compared with free Rh-B and Rh-B-loaded Cubs (Rh-B-Cubs). In vivo Rh-B-based tumor imaging demonstrated that Cubs-FA specifically targeted the tumor tissue. CONCLUSIONS The folate-modified cubosomes containing ETP may be a promising drug candidate for antitumor treatment.

Novel Strategy for Preparing Dual-Modality Optical/PET Imaging Probes via Photo-Click Chemistry.

Preparation of small molecule based dual-modality probes remains a challenging task due to the complicated synthetic procedure. In this study, a novel concise and generic strategy for preparing dual-modality optical/PET imaging probes via photo-click chemistry was developed, in which the diazole photo-click linker functioned not only as a bridge between the targeting-ligand and the PET imaging moiety, but also as the fluorophore for optical imaging. A dual-modality AE105 peptidic probe was successfully generated via this strategy and subsequently applied in the fluorescent staining of U87MG cells and the (68)Ga based PET imaging of mice bearing U87MG xenograft. In addition, dual-modality monoclonal antibody cetuximab has also been generated via this strategy and labeled with (64)Cu for PET imaging studies, broadening the application of this strategy to include the preparation of macromolecule based imaging probes.

Universal Molecular Scaffold for Facile Construction of Multivalent and Multimodal Imaging Probes.

Multivalent and multimodal imaging probes are rapidly emerging as powerful chemical tools for visualizing various biochemical processes. Herein, we described a bifunctional chelator (BFC)-based scaffold that can be used to construct such promising probes concisely. Compared to other reported similar scaffolds, this new BFC scaffold demonstrated two major advantages: (1) significantly simplified synthesis due to the use of this new BFC that can serve as chelator and linker simultaneously; (2) highly efficient synthesis rendered by using either click chemistry and/or total solid-phase synthesis. In addition, the versatile utility of this molecular scaffold has been demonstrated by constructing several multivalent/multimodal imaging probes labeled with various radioisotopes, and the resulting radiotracers demonstrated substantially improved in vivo performance compared to the two individual monomeric counterparts.

New Bifunctional Chelator p-SCN-PhPr-NE3TA for Copper-64: Synthesis, Peptidomimetic Conjugation, Radiolabeling, and Evaluation for PET Imaging.

Bifunctional chelators play an important role in developing metallic radionuclide-based radiopharmaceuticals. In this study, a new bifunctional ligand, p-SCN-PhPr-NE3TA, was synthesized and conjugated to a very late antigen-4 targeting peptidomimetic, LLP2A, for evaluating its application in (64)Cu-based positron emission tomography (PET) imaging. The new ligand exhibited strong selective coordination of Cu(II), leading to a robust Cu complex, even in the presence of 10-fold Fe(III). The LLP2A conjugate of p-SCN-PhPr-NE3TA was prepared and successfully labeled with (64)Cu under mild conditions. The conjugate (64)Cu-NE3TA-PEG4-LLP2A showed significantly higher specific activity, compared with (64)Cu-NOTA-PEG4-LLP2A, while maintaining comparable serum stability. Subsequent biodistribution studies and PET imaging in mice bearing B16F10 xenografts confirmed its favorable in vivo performance and high tumor uptake with low background, rendering p-SCN-PhPr-NE3TA a promising bifunctional chelator for (64)Cu-based radiopharmaceuticals.

Synthesis, anti-thymidine phosphorylase activity and molecular docking of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones.

In our lead finding program, a series of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones and their 5-thio-alkyl derivatives were designed and synthesized which contained different substituents at ortho-position of 2-phenyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the synthesized compounds exhibited a varying degree of inhibitory activity towards thymidine phosphorylase (TP), comparable to reference compound, 7-Deazaxanthine (7-DX, 2) (IC50 value=42.63 µM). The study also inferred that the ortho-substituted group at the phenyl ring and 5-thio-alkyl moiety imparted steric hindrance effects in the binding site of the enzyme, leading to a reduced inhibitory response. In addition, compound 3a was identified as a mixed-type inhibitor of TP. Moreover, computational docking study was performed to illustrate the important structural information on the plausible ligand-enzyme binding interactions.

Synthesis and evaluation of novel 1,4,7-triazacyclononane derivatives as Cu2+ and Ga3+ chelators.

Advances in chelator design are the cornerstone for the development of metals like copper and gallium based biomedical agents and radiopharmaceuticals. To develop optimal chelating ligands, we explored the synthesis and chelating properties of azaheterocycle pendant armed 1,4,7-triazacyclononane (TACN) dimethylcarboxylate derivatives and dimethylphosphonate derivatives. In the complexation kinetics test, dicarboxylate pendant armed TACN derivatives 2,2'-(7-((1H-imidazol-2-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA-Im), 2,2'-(7-((1-methyl-1H-imidazol-2-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA-MeIm), and 2,2'-(7-(thiazol-2-ylmethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA-Thia) exhibited fast complexation kinetics towards Cu (II) cations, which were comparable to the frequently explored ligand 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). And the diphosphonate pendant armed TACN derivative ((7-(thiazol-2-ylmethyl)-1,4,7-triazonane-1,4-diyl)bis(methylene))bis(phosphonic acid) (NODP-Thia) bound with Ga (III) cations at a much faster rate than NOTA. Density functional theory studies confirmed that the better complexation kinetics and metal chelating efficiency of NODA-Im, NODA-MeIm, NODA-Thia, and NODP-Thia could be ascribed to the lower Gibbs energies of corresponding chelator-metal complexes than NOTA-metal complexes. The kinetic inertness of the Cu (II) complex with NODA-Im, NODA-MeIm, and NODA-Thia was also demonstrated by cyclic voltammetry studies. Subsequently radiolabeling experiment demonstrated that these metal chelators could efficiently labeled with 64Cu or 68Ga in good radiochemical purities. These preliminary findings support NODA-Im, NODA-MeIm, NODA-Thia, and NODP-Thia as promising leading chelating agents for the development of bifunctional Cu2+ and Ga3+ chelators in biomedical applications.

Heterodimeric RGD-NGR PET Tracer for the Early Detection of Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal gastrointestinal cancer, and its poor prognosis is highly associated with the lack of an efficient early detection technology. Here, we report that RGD-NGR heterodimer labeled with PET isotope could be applied in PDAC early detection. PROCEDURES: The RGD-NGR tracer was first compared with its corresponding monomeric counterparts via PET imaging studies using mice bearing a subcutaneous BxPC3 tumor. Subsequently, the RGD-NGR tracer was evaluated in autochthonous mouse models with spontaneously developed late stage PanIN lesions (KCER mice) or PDAC (KPC mice) via both PET imaging studies and ex vivo biodistribution studies. Furthermore, a comparison between 2-deoxy-2[18F]fluoro-D-glucose ([18F]F-FDG) and the RGD-NGR tracer was conducted via PET imaging of the same KCH mouse bearing spontaneously developed PDAC. H&E staining was performed to confirm the malignant pancreatic tissue in the KCH mouse. Immunofluorescence staining was performed to confirm the expression of integrin αVß3 and CD13. RESULTS: The RGD-NGR tracer exhibited improved in vivo performance as compared with its corresponding monomeric counterparts on the subcutaneous BxPC3 tumor mouse model. Subsequent evaluation in autochthonous mouse models demonstrated its capability to detect both pre-malignant and malignant pancreases. Further comparison with [18F]F-FDG revealed the superiority of the proposed heterodimer in imaging spontaneously developed PDAC. H&E staining confirmed the malignant pancreatic tissue in the KCH mouse, while the expression of both integrin αVß3 and CD13 receptors was demonstrated with immunofluorescence staining. CONCLUSION: The proposed RGD-NGR heterodimer possesses the potential to be applied in the PDAC early detection for high-risk populations.

PET Imaging of CD8 via SMART for Monitoring the Immunotherapy Response.

Imaging of CD8 receptors on T-cells by positron emission tomography (PET) has been considered a promising strategy for monitoring the treatment response to immunotherapy. In this study, a trial of imaging CD8 with our newly developed sequential multiple-agent receptor targeting (SMART) technology was conducted. Mice bearing a subcutaneous colorectal CT26 tumor received three times different immunotherapy treatments (PD1 or CTLA4 or combined). On either day 7 or day 14 after the first time treatment, the PET imaging study was performed with sequentially administered TCO-modified anti-CD8 antibody and 64Cu-labeled MeTz-NOTA-RGD. However, no positive response was detected, probably due to (1) inappropriate selection of biomarkers for the SMART strategy, (2) limited TCO modification on the anti-CD8 antibody, and (3) inadequate response of the CT26 tumor to the selected immunotherapies. Therefore, the potential of applying SMART in imaging CD8 was not demonstrated in this study, and further optimization will be necessary before it can be applied in imaging CD8.

Development of Dual Receptor Enhanced Pre-Targeting Strategy-A Novel Promising Technology for Immuno-Positron Emission Tomography Imaging.

PET imaging has become an important diagnostic tool in the era of precise medicine. Various pre-targeting systems have been reported to address limitations associated with traditional immuno-PET. However, the application of these mono-receptor based pre-targeting (MRPT) strategies is limited to non-internalizable antibodies, and the tumor uptake is usually much lower than that in the corresponding immuno-PET. To circumvent these limitations, we develop the first Dual-Receptor Pre-Targeting (DRPT) system through entrapping the tumor-receptor-specific radioligand by the pre-administered antibody. Besides the similar ligation pathway happens in MRPT, incorporation of a tumor-receptor-specific peptide into the radioligand in DRPT enhances both concentration and retention of the radioligand on tumor, promoting its ligation with pre-administered mAb on cell-surface and/or internalized into tumor-cells. In this study, 64Cu based DRPT shows superior performance over corresponding MRPT and immuno-PET using internalizable antibodies. Besides, the compatibility of DRPT with short-lived and generator-produced 68Ga is demonstrated, leveraging its advantage in reducing radio-dose exposure. Furthermore, the feasibility of reducing the amount of the pre-administered antibody is confirmed, indicating the cost saving potential of DRPT. In summary, synergizing advantages of dual-receptor targeting and pre-targeting, we expect that this DRPT strategy can become a breakthrough technology in the field of antibody-based molecular imaging.

64Cu-Labeled Ubiquitin for PET Imaging of CXCR4 Expression in Mouse Breast Tumor.

Ubiquitin has been recently identified as a chemokine receptor 4 (CXCR4) natural ligand, offering great potential for positron emission computed tomography (PET) imaging of CXCR4 expression. This study reports the preparation and evaluation of (64Cu)-radiolabeled ubiquitin for CXCR4 imaging. The ubiquitin was first fused with a C-terminal GGCGG sequence, and the resulting recombinant ubiquitin derivative UbCG4 was then functionalized with the trans-cyclooctene (TCO) moiety via thiol-maleimide click reaction, followed by 64Cu-radiolabeling through the TCO/Tz (tetrazine)-based Diels-Alder click reaction. In the prepared in vitro studies, the prepared (64Cu)-UbCG4 showed significantly higher specific uptakes in the 4T1 breast cancer cells compared with the uptakes in the CXCR4-knockdown 4T1 cells. In the in vivo evaluation in the 4T1-xenograft mouse model, (64Cu)-UbCG4 demonstrated a similar tumor uptake but much lower backgrounds compared with 64Cu-labeled AMD3465. These results suggested that (64Cu)-UbCG4 could serve as a potent PET tracer for the noninvasive imaging of CXCR4 expression in tumors.

Dual-Targeted Molecular Imaging of Cancer.

Molecular imaging is critical to personalized and precision medicine. Although singly targeted imaging probes are making an impact both clinically and preclinically, molecular imaging strategies using bispecific probes have enabled improved visualization of cancer in recent years through synergistic targeting of two ligands. In this Focus on Molecular Imaging review, we outline how peptide-, antibody-, and nanoparticle-based platforms have affected this emerging strategy, providing examples and pointing out areas in which the greatest clinical impact may be realized.

A multi-functional polymeric carrier for simultaneous positron emission tomography imaging and combination therapy.

Multifunctional nanoplatforms offering simultaneous imaging and therapeutic functions have been recognized as a highly promising strategy for personalized nanomedicine. In this work, we synthesized a farnesylthiosalicylate (FTS, a nontoxic Ras antagonist) based triblock copolymer POEG-b-PVBA-b-PFTS (POVF) composed of a poly(oligo(ethylene glycol) methacrylate) (POEG) hydrophilic block, a poly(FTS) hydrophobic block, and a poly(4-vinylbenzyl azide) (PVBA) middle block. The POVF polymer itself was active in inhibiting the tumor growth in vitro and in vivo. Besides, it could serve as a carrier to effectively encapsulate paclitaxel (PTX) to form stable PTX/POVF mixed micelles with a diameter around 100 nm. Meanwhile, POVF polymer provides the active azide group for incorporating a positron emission tomography (PET) imaging modality via a facile strategy based on metal-free click chemistry. This nanocarrier system could not only be used for co-delivery of PTX and FTS, but also for PET imaging guided drug delivery. In the 4T1.2 tumor bearing mice, PET imaging showed rapid uptake and slow clearance of radiolabeled PTX/POVF nanomicelles in the tumor tissues. In addition, the FTS-based multi-functional nanocarrier was able to inhibit tumor growth effectively, and the co-delivery of PTX by the carrier further improved the therapeutic effect. STATEMENT OF SIGNIFICANCE: Due to the intrinsic heterogeneity of cancer and variability in individual patient response, personalized nanomedicine based on multi-functional carriers that integrate the functionalities of combination therapy and imaging guidance is highly demanded. Here we developed a multi-functional nanocarrier based on triblock copolymer POEG-b-PVBA-b-PFTS (POVF), which could not only be used for co-delivery of anticancer drugs PTX and Ras inhibitor FTS, but also for PET imaging guided drug delivery. The POVF carrier itself was active in inhibiting the tumor growth in vitro and in vivo. Besides, it was effective in formulating PTX with high drug loading capacity, which further enhanced the tumor inhibition effect. Meanwhile, we developed a simple and universal approach to incorporate a PET radioisotope (Zr-89 and Cu-64) into the azide-containing PTX/POVF micelles via metal-free click chemistry in aqueous solution. The radiolabeled PTX/POVF micelles exhibited excellent serum stability, rapid tumor uptake and slow clearance, which validated the feasibility of the PET image-guided delivery of PTX/POVF micelles.