Monitoring Therapeutic Responses to Silicified Cancer Cell Immunotherapy Using PET/MRI in a Mouse Model of Disseminated Ovarian Cancer

Current imaging approaches used to monitor tumor progression can lack the ability to distinguish true progression from pseudoprogression. Simultaneous metabolic 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI) offers new opportunities to overcome this challenge by refining tumor identification and monitoring therapeutic responses to cancer immunotherapy. In the current work, spatial and quantitative analysis of tumor burden were performed using simultaneous [¹⁸F]FDG-PET/MRI to monitor therapeutic responses to a novel silicified cancer cell immunotherapy in a mouse model of disseminated serous epithelial ovarian cancer. Tumor progression was validated by bioluminescence imaging of luciferase expressing tumor cells, flow cytometric analysis of immune cells in the tumor microenvironment, and histopathology. While PET demonstrated the presence of metabolically active cancer cells through [¹⁸F]FDG uptake, MRI confirmed cancer-related accumulation of ascites and tissue anatomy. This approach provides complementary information on disease status without a confounding signal from treatment-induced inflammation. This work provides a possible roadmap to facilitate accurate monitoring of therapeutic responses to cancer immunotherapies.     

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvβ3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [⁶⁸Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [⁶⁸Ga]Ga-DFO-c(RGDyK) can be used for αvβ3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.     

Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [^99mTc]Tc-(HE)₃-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)₃), provided clear imaging of HER2 expressing breast cancer 2–4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E₃C), Gly-Gly-Gly-Cys (G₃C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)₃-linker (designated as G3-(G₃S)₃C) would further improve the contrast of imaging using ^99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9–5 nM. Biodistribution of [^99mTc]Tc-G3-G₃C, [^99mTc]Tc-G3-(G₃S)₃C, and [^99mTc]Tc-G3-E₃C in mice was compared with the biodistribution of [^99mTc]Tc-(HE)₃-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [^99mTc]Tc-(HE)₃-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.     

Toward 18F-Labeled Theranostics: A Single Agent that Can Be Labeled with 18F, 64Cu,or 177Lu

We report a single-molecule radiotracer that can be labeled independently with ¹⁸F-fluoride or radiometals (⁶⁴Cu, ¹⁷⁷Lu) in a single step. A prostate-specific membrane antigen (PSMA)-targeting ligand, armed with both an organotrifluoroborate and a metal-chelator (DOTA), was designed to optionally afford ¹⁸F-, ⁶⁴Cu- or ¹⁷⁷Lu-labeled products that were injected into mice bearing prostate cancer (LNCaP) xenografts. PET/CT images and ex vivo biodistribution data show high, specific tumor uptake irrespective of which radionuclide is used, thereby demonstrating a new approach to combining, in a single molecule, ¹⁸F-labeling capabilities for PET imaging with radiometalation for potential imaging and therapeutic applications.     

Fully Automated Production and Characterization of 64Cu and Proof‐of‐Principle Small‐Animal PET Imaging Using 64Cu‐Labelled CA XII Targeting 6A10 Fab

⁶⁴Cu is a cyclotron‐produced radionuclide which offers, thanks to its characteristic decay scheme, the possibility of combining positron emission tomography (PET) investigations with radiotherapy. We evaluated the Alceo system from Comecer SpA to automatically produce ⁶⁴Cu for radiolabelling purposes. We established a ⁶⁴Cu production routine with high yields and radionuclide purity in combination with excellent operator radiation protection. The carbonic anhydrase XII targeting 6A10 antibody Fab fragment was successfully radiolabelled with the produced ⁶⁴Cu, and proof‐of‐principle small‐animal PET experiments on mice bearing glioma xenografts were performed. We obtained a high tumor‐to‐contralateral muscle ratio, which encourages further in vivo investigations of the radioconjugate regarding a possible application in diagnostic tumor imaging.     

A Pyropheophorbide Analogue Containing a Fused Methoxy Cyclohexenone Ring System Shows Promising Cancer-Imaging Ability

Herein we report the synthesis, photophysical properties, positron emission tomography (PET) imaging and photodynamic therapy (PDT) efficacy of methyl 3-(1′-m-iodobenzyloxy)ethyl-3-devinyl-verdin 4 (with or without the ¹²⁴I isotope). The PET imaging ability and ex vivo biodistribution of [¹²⁴I] 4 were compared with the well-studied methyl [3-(¹²⁴1′-m-iodobenzyloxy)ethyl]-3-devinyl-pyropheophorbide-a methyl ester (PET-ONCO or [¹²⁴I] 2 ) and [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) in BALB/c mice bearing colon-26 tumors. Whole-body PET images of [¹²⁴I] 4 containing a fused methoxy cyclohexenone ring system showed excellent tumor contrast with time (72>48>24 h post-injection). Ex vivo biodistribution results indicate that relative to the current clinical standard [¹⁸F]FDG and [¹²⁴I] 2 in 2 % ethanol formulation, [¹²⁴I] 4 , at the same radioactive dose (25 μCi per mouse), showed higher tumor uptake at 24 h post-injection and longer tumor retention. In biological environments, compound 4 showed lower fluorescence and lower singlet oxygen yield than 2 , which is possibly due to higher aggregation caused by the presence of a fused cyclohexenone ring system, resulting in limited in vitro/in vivo PDT efficacy. Therefore, the chlorophyll-a analogue [¹²⁴I] 4 provides easy access to a novel PET imaging agent (with no skin phototoxicity) to image cancer types—brain, renal carcinomas, pancreas—in which [¹⁸F]FDG shows limitations.     

Anticancer Gold(III) Peptidomimetics: From Synthesis to in vitro and ex vivo Biological Evaluations

Five new Au^III‐peptidodithiocarbamato complexes of the type [Au^IIIBr₂(dtc‐AA₁‐AA₂‐OR] (in which AA₁=N‐methylglycine (Sar), l /d ‐Pro; AA₂=l /d ‐Ala, α‐aminoisobutyric acid (Aib); R=OtBu, triethylene glycol methyl ether), differing with regard to the amino acid sequence and/or the chiral amino acid configuration, were designed to enhance tumor selectivity and bioavailability. The gold(III)‐based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, one‐ and two‐dimensional NMR spectroscopy, FT‐IR, and UV/Vis spectrophotometry. The crystal structures of three compounds were also solved by X‐ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549 [non‐small‐cell lung carcinoma], MCF‐7 [breast cancer], A2780 [ovarian carcinoma], H1975 [non‐small‐cell lung carcinoma], H460 [large‐cell lung carcinoma], and A431 [human epidermoid carcinoma]) showed the dtc‐Pro‐Aib‐OtBu derivative to be very effective, with GI₅₀ values much lower than those of cisplatin. This complex was thus selected for evaluating stability under physiological conditions and possible interactions with serum albumin, as well in PARP‐1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues.     

Synthesis and Anticancer Activity of Tertiary Amides of Salinomycin and Their C20-oxo Analogues

The polyether ionophore salinomycin ( SAL ) has captured much interest because of its potent activity against cancer cells and cancer stem cells. Our previous studies have indicated that C1/C20 double-modification of SAL is a useful strategy to generate diverse agents with promising biological activity profiles. Thus, herein we describe the synthesis of a new class of SAL analogues that combine key modifications at the C1 and C20 positions. The activity of the obtained SAL derivatives was evaluated using primary acute lymphoblastic leukemia, human breast adenocarcinoma and normal mammary epithelial cells. One single- [N,N-dipropyl amide of salinomycin ( 5 a )] and two novel double-modified analogues [N,N-dipropyl amide of C20-oxosalinomycin ( 5 b ) and piperazine amide of C20-oxosalinomycin ( 13 b )] were found to be more potent toward the MDA-MB-231 cell line than SAL or its C20-oxo analogue 2 . When select analogues were tested against the NCI-60 human tumor cell line panel, 4 a [N,N-diethyl amide of salinomycin] showed particular activity toward the ovarian cancer cell line SK-OV-3. Additionally, both SAL and 2 were found to be potent ex vivo against human ER/PR⁺, Her2⁻ invasive mammary carcinoma, with 2 showing minimal toxicity toward normal epithelial cells. The present findings highlight the therapeutic potential of SAL derivatives for select targeting of different cancer types.     

Comparative Assessment of Complex Stabilities of Radiocopper Chelating Agents by a Combination of Complex Challenge and in vivo Experiments

For ⁶⁴Cu radiolabeling of biomolecules to be used as in vivo positron emission tomography (PET) imaging agents, various chelators are commonly applied. It has not yet been determined which of the most potent chelators—NODA‐GA ((1,4,7‐triazacyclononane‐4,7‐diyl)diacetic acid‐1‐glutaric acid), CB‐TE2A (2,2′‐(1,4,8,11‐tetraazabicyclo[6.6.2]hexadecane‐4,11‐diyl)diacetic acid), or CB‐TE1A‐GA (1,4,8,11‐tetraazabicyclo[6.6.2]hexadecane‐4,11‐diyl‐8‐acetic acid‐1‐glutaric acid)—forms the most stable complexes resulting in PET images of highest quality. We determined the ⁶⁴Cu complex stabilities for these three chelators by a combination of complex challenge and an in vivo approach. For this purpose, bioconjugates of the chelating agents with the gastrin‐releasing peptide receptor (GRPR)‐affine peptide PESIN and an integrin α_vβ₃‐affine c(RGDfC) tetramer were synthesized and radiolabeled with ⁶⁴Cu in excellent yields and specific activities. The ⁶⁴Cu‐labeled biomolecules were evaluated for their complex stabilities in vitro by conducting a challenge experiment with the respective other chelators as challengers. The in vivo stabilities of the complexes were also determined, showing the highest stability for the ⁶⁴Cu–CB‐TE1A‐GA complex in both experimental setups. Therefore, CB‐TE1A‐GA is the most appropriate chelating agent for *Cu‐labeled radiotracers and in vivo imaging applications.     

Design,Synthesis and In Vivo Fluorescence Imaging Study of a Cytochrome P450 1B1 Targeted NIR Probe Containing a Chelator Moiety

Cytochrome P450 (CYP) 1B1 has been found to be overexpressed specifically in tumor tissues at an early stage, which makes it a potential cancer biomarker for molecular imaging. Multimodal imaging combines different imaging modalities and offers more comprehensive information. Thus, imaging probes bearing more than one kind of signal fragment have been extensively explored and display great promise. Herein, we developed a near infrared (NIR) probe with a chelator moiety targeting CYP1B1 by conjugating α-naphthoflavone (ANF) derivatives with both an NIR dye and a chelator for potential application in bimodal imaging. Enzymatic inhibitory studies demonstrated inhibitory activity against CYP1B1 and selectivity among CYP1 were successfully retained after chemical modification. Cell-based saturation studies indicated nanomolar range binding affinity between the probe and CYP1B1 overexpressed cancer cells. In vitro competitive binding assays monitored by confocal microscopy revealed that the probe could specifically accumulate in tumor cells. In vivo and ex vivo imaging studies demonstrated that the probe could effectively light-up the tumor tissues as early as 2 hours post-injection. In addition, the fluorescence was significantly blocked by co-injection of CYP1B1 inhibitor, which indicated the probe accumulation in tumor sites was due to specific binding to CYP1B1.     

TAMpepK Suppresses Metastasis through the Elimination of M2-Like Tumor-Associated Macrophages in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all breast cancer cases and is characterized by high invasiveness, high metastatic potential, relapse proneness, and poor prognosis. M2-like tumor-associated macrophages (TAMs) contribute to tumorigenesis and are promising targets for inhibiting breast cancer metastasis. Therefore, we investigated whether melittin-conjugated pro-apoptotic peptide (TAMpepK) exerts therapeutic effects on breast cancer metastasis by targeting M2-like TAMs. TAMpepK is composed of M2-like TAM binding peptide (TAMpep) and pro-apoptotic peptide d(KLAKLAK)₂ (dKLA). A metastatic mouse model was constructed by injecting 4T1-luc2 cells either orthotopically or via tail vein injection, and tumor burden was quantified using a bioluminescence in vivo imaging system. We found that TAMpepK suppressed lung and lymph node metastases of breast cancer by eliminating M2-like TAMs without affecting the viability of M1-like macrophages and resident macrophages in the orthotopic model. Furthermore, TAMpepK reduced pulmonary seeding and the colonization of tumor cells in the tail vein injection model. The number of CD8⁺ T cells in contact with TAMs was significantly decreased in tumor nodules treated with TAMpepK, resulting in the functional activation of cytotoxic CD8⁺ T cells. Taken together, our findings suggest that TAMpepK could be a novel therapeutic agent for the inhibition of breast cancer metastasis by targeting M2-like TAMs.     

Head-to-Head Comparison of Fibroblast Activation Protein Inhibitors (FAPI) Radiotracers versus [18F]F-FDG in Oncology: A Systematic Review

Several recent studies comparing radiolabeled fibroblast activation protein inhibitors (FAPI) and fluorine-18 fluorodeoxyglucose ([¹⁸F]F-FDG) as positron emission tomography (PET) radiotracers in oncology have been published. The aim of this systematic review is to perform an updated evidence-based summary about the comparison of these PET radiotracers in oncology to better address further research in this setting. Studies or subsets of studies comparing radiolabeled FAPI and [¹⁸F]F-FDG as PET radiotracers in oncology were eligible for inclusion in this systematic review. A systematic literature search of PubMed/MEDLINE and Cochrane library databases was performed until August 2021. Literature data about the comparison of [¹⁸F]F-FDG and radiolabeled FAPI are rapidly increasing. Overall, taking into account radiotracer uptake and tumor-to-background uptake ratio, compared to [¹⁸F]F-FDG PET, an equal or higher detection of primary tumors and/or metastatic lesions was usually demonstrated by using radiolabeled FAPI PET. In particular, the cancer entities with better detection rate of tumor lesions by using radiolabeled FAPI PET, compared to [¹⁸F]F-FDG PET, were gastrointestinal tumors, liver tumors, breast cancer and nasopharyngeal carcinoma. Further comparison studies are needed to better evaluate the best field of application of radiolabeled FAPI PET.     

Design,Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC₅₀ of 0.53±0.8 μM in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [¹⁸F] 3b was successfully developed. Unfortunately, the stability of [¹⁸F] 3b turned out to be insufficient to pursue imaging studies.     

Fluorescent magnetic nanoparticle-labeled mesenchymal stem cells for targeted imaging and hyperthermia therapy of in vivo gastric cancer

How to find early gastric cancer cells in vivo is a great challenge for the diagnosis and therapy of gastric cancer. This study is aimed at investigating the feasibility of using fluorescent magnetic nanoparticle (FMNP)-labeled mesenchymal stem cells (MSCs) to realize targeted imaging and hyperthermia therapy of in vivo gastric cancer. The primary cultured mouse marrow MSCs were labeled with amino-modified FMNPs then intravenously injected into mouse model with subcutaneous gastric tumor, and then, the in vivo distribution of FMNP-labeled MSCs was observed by using fluorescence imaging system and magnetic resonance imaging system. After FMNP-labeled MSCs arrived in local tumor tissues, subcutaneous tumor tissues in nude mice were treated under external alternating magnetic field. The possible mechanism of MSCs targeting gastric cancer was investigated by using a micro-multiwell chemotaxis chamber assay. Results show that MSCs were labeled with FMNPs efficiently and kept stable fluorescent signal and magnetic properties within 14 days, FMNP-labeled MSCs could target and image in vivo gastric cancer cells after being intravenously injected for 14 days, FMNP-labeled MSCs could significantly inhibit the growth of in vivo gastric cancer because of hyperthermia effects, and CCL19/CCR7 and CXCL12/CXCR4 axis loops may play key roles in the targeting of MSCs to in vivo gastric cancer. In conclusion, FMNP-labeled MSCs could target in vivo gastric cancer cells and have great potential in applications such as imaging, diagnosis, and hyperthermia therapy of early gastric cancer in the near future.     

Synthesis and characterization of poly(HPMA)‐APMA‐DTPA‐99mTc for imaging‐guided drug delivery in hepatocellular carcinoma

To develop a theranostic agent for diagnostic imaging and treatment of  hepatocellular carcinoma (HCC), poly(HPMA)‐APMA‐DTPA‐^99mTc (HPMA: N‐(2‐hydroxypropyl methacrylamide; APMA: N‐(3‐aminopropyl)methacrylamide; DTPA: diethylenetriaminepentaacetic acid) and DTPA‐^99mTc were synthesized and characterized, and their HCC targeting was tested by in vitro cellular uptake and in vivo tumor imaging in this study. Radioactivity of HCC cells incubated with poly(HPMA)‐APMA‐DTPA‐^99mTc was significant higher (16.40%) than that of the cells incubated with DTPA‐^99mTc (2.98%). Scintigraphic images of HCC in mice obtained at 8 h after injection of poly(HPMA)‐APMA‐DTPA‐^99mTc showed increased radioactivity compared with that in mice injected with DTPA‐^99mTc. The results of postmortem tissue radioactivity assay demonstrated higher radioactivity of HCC tumor tissues (2.69 ± 0.15% ID/g) from the tumor‐bearing mice injected with poly(HPMA)‐APMA‐DTPA‐^99mTc compared with that of HCC tumor tissues in the tumor‐bearing mice injected with DTPA‐^99mTc (0.83 ± 0.03 %ID/g), (P <0.001). These results first directly confirm the significant passive hepatocellular tumor targeting of HPMA copolymer. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Action of the new hypolipidemic agent lifibrol (K12.148) on lipid homeostasis in normal rats: Plasma lipids,hepatic sterologenesis,and the fate of injected [14C] acetate

Lifibrol, a new hypocholesterolemic agent with activity in humans, was examined in normal rats for its short-term and long-term effects on lipid homeostasis. Cholesterol (Chol) synthesis inhibition by lifibrol was demonstratedin vitro in liver minces from normal rats by following [1-¹⁴C]acetate ([¹⁴C]Ac) and DL-[2-¹⁴C]mevalonate ([¹⁴C]-MVA) incorporation into [¹⁴C]Chol. When administered at 50 mg/kg/d, lifibrol reduced plasma total Chol and triglycerides (TG) (P<0.001) within 24 h. The Chol reduction was largely a result of reduction of low density and very low density lipoprotein cholesterol (LDL+VLDL-chol) and a smaller decrease in high density lipoprotein cholesterol (HDL-chol). After 10 d, however, a rebound effect emerged, and after 41 d, plasma Chol, LDL+VLDL-chol, and HDL-chol were restored. In contrast, plasma TG remained at reduced levels (P<0.01). The rebound is attributed to counter-regulation of hepatic sterologenesis that was assessed bothex vivo andin vivo. Theex vivo incorporation of [¹⁴C]MVA and [¹⁴C]octanoate into [¹⁴C]Chol and total digitonin-precipitable [¹⁴C]sterols ([¹⁴C]DPS) in liver minces was increased 2-and 6-fold, respectively, in rats treated 6 d at 50 mg/kg. Similarly,in vivo incorporation of intraperitoneally injected [¹⁴C]Ac into hepatic [¹⁴C]DPS (2 h post-injection) was increased 2- to 5-fold at 50 mg/kg, and evidence for increased sterologenesis in nonhepatic tissue was also obtained. The increased hepatic sterologenesis, evident within 48 h, persisted out to 41 d of treatment by which time increases (P<0.002) in hepatic Chol and carcass total sterols were observed. Additionally, incorporation of injected [¹⁴C]Ac into hepatic [¹⁴C]TG was inhibited 60% by lifibrol (P<0.001), and the appearance of [¹⁴C]TG in plasma was reduced. Circulating free [¹⁴C]fatty acids ([¹⁴C]FFA) were also reduced, but hepatic [¹⁴C]FFA synthesis was unaffected, thus suggesting either a lesser release of newly formed FFA from liver or an enhanced removal from plasma.     

Room Temperature Al18F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging

Positron emission tomography (PET) is a non-invasive molecular imaging technology that is constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i. e. Fab, svFab, nanobodies) is increasing and has led us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which were investigated as such for {Al¹⁸F}²⁺-core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed under various conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240 min in human serum (HS), EDTA 5 mM, PBS and 0.9 % NaCl solutions. The in vivo stability of [Al¹⁸F(2-AMPDA-HB)]⁻ was assessed in healthy nude mice (n=6). Radiochemical yields between 55 % and 81 % were obtained at pH 5 and room temperature. High stability in HS was measured for [Al¹⁸F(2-AMPDA-HB)]⁻, with 90 % of F-18 complexed after 120 min. High stability in vivo, rapid hepatobiliary and renal excretion, with low accumulation of free F-18 in bones were measured. Thus, this new Al¹⁸F-chelator may have a great impact on immuno-PET radiopharmacy, by facilitating the development of new fluorine-18-labeled heat-sensitive biomolecules.     

Theranostic Small-Molecule Prodrug Conjugates for Targeted Delivery and Controlled Release of Toll-like Receptor 7 Agonists

We previously reported the design and synthesis of a small-molecule drug conjugate (SMDC) platform that demonstrated several advantages over antibody–drug conjugates (ADCs) in terms of in vivo pharmacokinetics, solid tumor penetration, definitive chemical structure, and adaptability for modular synthesis. Constructed on a tri-modal SMDC platform derived from 1,3,5-triazine (TZ) that consists of a targeting moiety (Lys-Urea-Glu) for prostate-specific membrane antigen (PSMA), here we report a novel class of chemically identical theranostic small-molecule prodrug conjugates (T-SMPDCs), [^18/19F]F-TZ(PSMA)-LEGU-TLR7, for PSMA-targeted delivery and controlled release of toll-like receptor 7 (TLR7) agonists to elicit de novo immune response for cancer immunotherapy. In vitro competitive binding assay of [¹⁹F]F-TZ(PSMA)-LEGU-TLR7 showed that the chemical modification of Lys-Urea-Glu did not compromise its binding affinity to PSMA. Receptor-mediated cell internalization upon the PSMA binding of [¹⁸F]F-TZ(PSMA)-LEGU-TLR7 showed a time-dependent increase, indicative of targeted intracellular delivery of the theranostic prodrug conjugate. The designed controlled release of gardiquimod, a TLR7 agonist, was realized by a legumain cleavable linker. We further performed an in vivo PET/CT imaging study that showed significantly higher uptake of [¹⁸F]F-TZ(PSMA)-LEGU-TLR7 in PSMA⁺ PC3-PIP tumors (1.9 ± 0.4% ID/g) than in PSMA⁻ PC3-Flu tumors (0.8 ± 0.3% ID/g) at 1 h post-injection. In addition, the conjugate showed a one-compartment kinetic profile and in vivo stability. Taken together, our proof-of-concept biological evaluation demonstrated the potential of our T-SMPDCs for cancer immunomodulatory therapies.     

Systemic Delivery of Protein Nanocages Bearing CTT Peptides for Enhanced Imaging of MMP-2 Expression in Metastatic Tumor Models

Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.     

Synthesis of a Bifunctional Cross-Bridged Chelating Agent,Peptide Conjugation,and Comparison of 68Ga Labeling and Complex Stability Characteristics with Established Chelators

[⁶⁸Ga]Ga³⁺ can be introduced into receptor-specific peptidic carriers via different chelators to obtain radiotracers for Positron Emission Tomography imaging and the chosen chelating agent considerably influences the in vivo pharmacokinetics of the corresponding radiopeptides. A chelator that should be a valuable alternative to established chelating agents for ⁶⁸Ga-radiolabeling of peptides would be a backbone-functionalized variant of the chelator CB-DO2A. Here, the bifunctional cross-bridged chelating agent CB-DO2A-GA was developed and compared to the established chelators DOTA, NODA-GA and DOTA-GA. For this purpose, CB-DO2A-GA(tBu)₂ was introduced into the peptide Tyr³-octreotate (TATE) and in direct comparison to the corresponding DOTA-, NODA-GA-, and DOTA-GA-modified TATE analogs, CB-DO2A-GA-TATE required harsher reaction conditions for ⁶⁸Ga-incorporation. Regarding the hydrophilicity profile of the resulting radiopeptides, a decrease in hydrophilicity from [⁶⁸Ga]Ga-DOTA-GA-TATE (log_D(7.4) of −4.11±0.11) to [⁶⁸Ga]Ga-CB-DO2A-GA-TATE (−3.02±0.08) was observed. Assessing the stability against metabolic degradation and complex challenge, [⁶⁸Ga]Ga-CB-DO2A-GA demonstrated a very high kinetic inertness, exceeding that of [⁶⁸Ga]Ga-DOTA-GA. Therefore, CB-DO2A-GA is a valuable alternative to established chelating agents for ⁶⁸Ga-radiolabeling of peptides, especially when the formation of a very stable, positively charged ⁶⁸Ga-complex is pursued.