Accuracy of gross tumour volume delineation with [68Ga]-PSMA-PET compared to histopathology for high-risk prostate cancer.

BACKGROUND: The delineation of intraprostatic lesions is vital for correct delivery of focal radiotherapy boost in patients with prostate cancer (PC). Errors in the delineation could translate into reduced tumour control and potentially increase the side effects. The purpose of this study is to compare PET-based delineation methods with histopathology. MATERIALS AND METHODS: The study population consisted of 15 patients with confirmed high-risk PC intended for prostatectomy. [68Ga]-PSMA-PET/MR was performed prior to surgery. Prostate lesions identified in histopathology were transferred to the in vivo [68Ga]-PSMA-PET/MR coordinate system. Four radiation oncologists manually delineated intraprostatic lesions based on PET data. Various semi-automatic segmentation methods were employed, including absolute and relative thresholds, adaptive threshold, and multi-level Otsu threshold. RESULTS: The gross tumour volumes (GTVs) delineated by the oncologists showed a moderate level of interobserver agreement with Dice similarity coefficient (DSC) of 0.68. In comparison with histopathology, manual delineations exhibited the highest median DSC and the lowest false discovery rate (FDR) among all approaches. Among semi-automatic approaches, GTVs generated using standardized uptake value (SUV) thresholds above 4 (SUV > 4) demonstrated the highest median DSC (0.41), with 0.51 median lesion coverage ratio, FDR of 0.66 and the 95th percentile of the Hausdorff distance (HD95%) of 8.22 mm. INTERPRETATION: Manual delineations showed a moderate level of interobserver agreement. Compared to histopathology, manual delineations and SUV > 4 exhibited the highest DSC and the lowest HD95% values. The methods that resulted in a high lesion coverage were associated with a large overestimation of the size of the lesions.

Moving Beyond Isothiocyanates: A Look at the Stability of Conjugation Links Toward Radiolysis in 89Zr-Labeled Immunoconjugates.

Zirconium-89 is the most widely used radioisotope for immunoPET because its physical half-life (78.2 h) suits the one of antibodies. Desferrioxamine B (DFO) is the standard chelator for the complexation of zirconium(IV), and its bifunctional version, containing a phenylisothiocyanate function, is the most commonly used for the conjugation of DFO to proteins. However, preliminary results have shown that the thiourea link obtained from the conjugation of isothiocyanate and lysines is sensitive to the ionizing radiation generated by the radioisotope, leading to the rupture of the link and the release of the chelator/radiometal complex. This radiolysis phenomenon could produce nonspecific signal and prevent the detection of bone metastasis, as free zirconium accumulates into the bones. The aim of this work was to study the stability of a selection of conjugation linkers in 89Zr-labeled immunoconjugates. We have synthesized several DFO-based bifunctional chelators appended with an isothiocyanate moiety, a bicyclononyne, or a squaramate ester. Two antibodies (trastuzumab and rituximab) were conjugated and radiolabeled with zirconium-89. The effect of increasing activities of zirconium-89 on the integrity of the bioconjugate bearing thiourea links was evaluated as well as the impact of the presence of a radioprotectant. The stability of the radiolabeled antibodies was studied over 7 days in PBS and human plasma. Radioconjugates' integrity was evaluated using iTLC and size-exclusion chromatography. This study shows that the nature of the linker between the chelator and biomolecule can have a strong impact on the stability of the 89Zr-labeled conjugates, as well as on the aggregation of the conjugates.

Development of an Immuno-SPECT/Fluorescent Bimodal Tracer Targeting Human or Murine PD-L1 on Preclinical Models.

Detection of biomarkers to diagnose, treat, and predict the efficacy of cancer therapies is a major clinical challenge. Currently, biomarkers such as PD-L1 are commonly detected from biopsies, but this approach does not take into account the spatiotemporal heterogeneity of their expression in tumors. A solution consists in conjugating monoclonal antibodies (mAbs) targeting these biomarkers with multimodal imaging probes. In this study, a bimodal [111In]-DOTA-aza-BODIPY probe emitting in the near-infrared (NIR) was grafted onto mAbs targeting murine or human PD-L1 either in a site-specific or random manner. In vitro, these bimodal mAbs showed a good stability and affinity for PD-L1. In vivo, they targeted specifically PD-L1 and were detected by both fluorescence and SPECT imaging. A significant benefit of site-specific conjugation on glycans was observed compared to random conjugation on lysine. The potential of this bimodal agent was also highlighted, thanks to a proof of concept of fluorescence-guided surgery in a human PD-L1+ tumor model.

Cherenkov Radiation induced photodynamic therapy - repurposing older photosensitizers, and radionuclides.

CONTEXT: Old-generation photosensitizers are minimally used in current photodynamic therapy (PDT) because they absorb in the UV/blue/green region of the spectrum where biological tissues are generally highly absorbing. The UV/blue light of Cherenkov Radiation (CR) from nuclear disintegration of beta-emitter radionuclides shows promise as an internal light source to activate these photosensitizers within tissue. Outline of the study: 1) radionuclide choice and Cherenkov Radiation, 2) Photosensitizer choice, synthesis and radiolabeling, 3) CR-induced fluorescence, 4) Verification of ROS formation, 5) CR-induced PDT with either free eosine and free CR emitter, or with radiolabelled eosin. RESULTS: Cherenkov Radiation Energy Transfer (CRET) from therapeutic radionuclides (90Y) and PET imaging radionuclides (18F, 68Ga) to eosin was shown by spectrofluorimetry and in vitro, and was shown to result in a PDT process. The feasibility of CR-induced PDT (CR-PDT) was demonstrated in vitro on B16F10 murine melanoma cells mixing free eosin (λabs = 524 nm, ΦΔ 0.67) with free CR-emitter [18F]-FDG under their respective intrinsic toxicity levels (0.5 mM/8 MBq) and by trapping singlet oxygen with diphenylisobenzofuran (DPBF). An eosin-DOTAGA-chelate conjugate 1 was synthesized and radiometallated with CR-emitter [68Ga] allowed to reach 25 % cell toxicity at 0.125 mM/2 MBq, i.e. below the toxicity threshold of each component measured on controls. Incubation time was carefully examined, especially for CR emitters, in light of its toxicity, and its CR-emitting yield expected to be 3 times as much for 68Ga than 18F (considering their ß particle energy) per radionuclide decay, while its half-life is about twice as small. PERSPECTIVE: This study showed that in complete darkness, as it is at depth in tissues, PDT could proceed relying on CR emission from radionuclides only. Interestingly, this study also repurposed PET imaging radionuclides, such as 68Ga, to trigger a therapeutic event (PDT), albeit in a modest extent. Moreover, although it remains modest, such a PDT approach may be used to achieve additional tumoricidal effect to RIT treatment, where radionuclides, such as 90Y, are strong CR emitters, i.e. very potent light source for photosensitizer activation.

SPECT Imaging of Lysyl Oxidase-like 2 in a Model of Idiopathic Pulmonary Fibrosis.

Noninvasive imaging of idiopathic pulmonary fibrosis (IPF) remains a challenge. The aim of this study was to develop an antibody-based radiotracer targeting Lysyl Oxidase-like 2 (LOXL2), an enzyme involved in the fibrogenesis process, for SPECT/CT imaging of pulmonary fibrosis. The bifunctional chelator DOTAGA-PEG4-NH2 was chemoenzymatically conjugated to the murine antibody AB0023 using microbial transglutaminase, resulting in a degree of labeling (number of chelators per antibody) of 2.3. Biolayer interferometry confirmed that the binding affinity of DOTAGA-AB0023 to LOXL2 was preserved with a dissociation constant of 2.45 ± 0.04 nM. DOTAGA-AB0023 was then labeled with 111In and in vivo experiments were carried out in a mice model of progressive pulmonary fibrosis induced by intratracheal administration of bleomycin. [111In]In-DOTAGA-AB0023 was injected in three groups of mice (control, fibrotic, and treated with nintedanib). SPECT/CT images were recorded over 4 days p.i. and an ex vivo biodistribution study was performed by gamma counting. A significant accumulation of the tracer in the lungs of the fibrotic mice was observed at D18 post-bleomycin. Interestingly, the tracer uptake was found selectively upregulated in fibrotic lesions observed on CT scans. Images of mice that received the antifibrotic drug nintedanib from D8 up to D18 showed a decrease in [111In]In-DOTAGA-AB0023 lung uptake associated with a decrease in pulmonary fibrosis measured by CT scan. In conclusion, we report the first radioimmunotracer targeting the protein LOXL2 for nuclear imaging of IPF. The tracer showed promising results in a preclinical model of bleomycin-induced pulmonary fibrosis, with high lung uptake in fibrotic areas, and accounted for the antifibrotic activity of nintedanib.

First Comparison Study of the In Vitro and In Vivo Properties of a Randomly and Site-Specifically Conjugated SPECT/NIRF Monomolecular Multimodal Imaging Probe (MOMIP) Based on an aza-BODIPY Fluorophore.

Among all approaches in molecular imaging, the combination of near-infrared fluorescence imaging (NIRF) with radioisotopic imaging (PET or SPECT) allows one to benefit from the advantages of each of the imaging techniques, which are very complementary and of comparable sensitivity. To this end, the construction of monomolecular multimodal probes (MOMIP) has made it possible to combine the two imaging modalities within the same molecule, thus limiting the number of bioconjugation sites and yielding more homogeneous conjugates compared with those prepared through sequential conjugation. However, in order to optimize the bioconjugation strategy and, at the same time, the pharmacokinetic and biodistribution properties of the resulting imaging agent, a site-specific approach may be preferred. To further investigate this hypothesis, random and glycan-based site-specific bioconjugation approaches were compared thanks to a SPECT/NIRF bimodal probe based on an aza-BODIPY fluorophore. The overall experiments conducted in vitro and in vivo on HER2-expressing tumors demonstrated a clear superiority of the site-specific approach to improve affinity, specificity, and biodistribution of the bioconjugates.

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors.

Because positron emission tomography (PET) and optical imaging are very complementary, the combination of these two imaging modalities is very enticing in the oncology field. Such bimodal imaging generally relies on imaging agents bearing two different imaging reporters. In the bioconjugation field, this is mainly performed by successive random conjugations of the two reporters on the protein vector, but these random conjugations can alter the vector properties. In this study, we aimed at abrogating the heterogeneity of the bimodal imaging immunoconjugate and mitigating the impact of multiple random conjugations. A trivalent platform bearing a DFO chelator for 89Zr labeling, a NIR fluorophore, IRDye800CW, and a bioconjugation handle was synthesized. This bimodal probe was site-specifically grafted to trastuzumab via glycan engineering. This new bimodal immunoconjugate was then investigated in terms of radiochemistry, in vitro and in vivo, and compared to the clinically relevant random equivalent. In vitro and in vivo, our strategy provides several improvements over the current clinical standard. The combination of site-specific conjugation with the monomolecular platform reduced the heterogeneity of the final immunoconjugate, improved the resistance of the fluorophore toward radiobleaching, and reduced the nonspecific uptake in the spleen and liver compared to the standard random immunoconjugate. To conclude, the strategy developed is very promising for the synthesis of better defined dual-labeled immunoconjugates, although there is still room for improvement. Importantly, this conjugation strategy is highly modular and could be used for the synthesis of a wide range of dual-labeled immunoconjugates.

The GRP94 Inhibitor PU-WS13 Decreases M2-like Macrophages in Murine TNBC Tumors: A Pharmaco-Imaging Study with 99mTc-Tilmanocept SPECT.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and is not eligible for hormone and anti-HER2 therapies. Identifying therapeutic targets and associated biomarkers in TNBC is a clinical challenge to improve patients' outcome and management. High infiltration of CD206+ M2-like macrophages in the tumor microenvironment (TME) indicates poor prognosis and survival in TNBC patients. As we previously showed that membrane expression of GRP94, an endoplasmic reticulum chaperone, was associated with the anti-inflammatory profile of human PBMC-derived M2 macrophages, we hypothesized that intra-tumoral CD206+ M2 macrophages expressing GRP94 may represent innovative targets in TNBC for theranostic purposes. We demonstrate in a preclinical model of 4T1 breast tumor-bearing BALB/c mice that (i) CD206-expressing M2-like macrophages in the TME of TNBC can be specifically detected and quantified using in vivo SPECT imaging with 99mTc-Tilmanocept, and (ii) the inhibition of GRP94 with the chemical inhibitor PU-WS13 induces a decrease in CD206-expressing M2-like macrophages in TME. This result correlated with reduced tumor growth and collagen content, as well as an increase in CD8+ cells in the TME. 99mTc-Tilmanocept SPECT imaging might represent an innovative non-invasive strategy to quantify CD206+ tumor-associated macrophages as a biomarker of anti-GRP94 therapy efficacy and TNBC tumor aggressiveness.

Development of an Easily Bioconjugatable Water-Soluble Single-Photon Emission-Computed Tomography/Optical Imaging Bimodal Imaging Probe Based on the aza-BODIPY Fluorophore.

A water-soluble fluorescent aza-BODIPY platform (Wazaby) was prepared and functionalized by a polyazamacrocycle agent and a bioconjugable arm. The resulting fluorescent derivative was characterized and bioconjugated onto a trastuzumab monoclonal antibody as a vector. After bioconjugation, the imaging agent appeared to be stable in serum (>72 h at 37 °C) and specifically labeled HER-2-positive breast tumors slices. The bioconjugate was radiolabeled with [111In] indium and studied in vivo. The developed monomolecular multimodal imaging probe (MOMIP) is water-soluble and chemically and photochemically stable, emits in the near infrared (NIR) region (734 nm in aqueous media), and displays a good quantum yield of fluorescence (around 15%). Single-photon emission-computed tomography and fluorescence imaging have been performed in nude mice bearing HER2-overexpressing HCC1954 human breast cancer xenografts and have evidenced the good tumor targeting of the [111In] In bimodal agent. Finally, the proof of concept of using it as a new tool for fluorescence-guided surgery has been shown.

Positron Emission Tomography Imaging of Neurotensin Receptor-Positive Tumors with 68Ga-Labeled Antagonists: The Chelate Makes the Difference Again.

Neurotensin receptor 1 (NTS1) is involved in the development and progression of numerous cancers, which makes it an interesting target for the development of diagnostic and therapeutic agents. A small molecule NTS1 antagonist, named [177Lu]Lu-IPN01087, is currently evaluated in phase I/II clinical trials for the targeted therapy of neurotensin receptor-positive cancers. In this study, we synthesized seven compounds based on the structure of NTS1 antagonists, bearing different chelating agents, and radiolabeled them with gallium-68 for PET imaging. These compounds were evaluated in vitro and in vivo in mice bearing a HT-29 xenograft. The compound [68Ga]Ga-bisNODAGA-16 showed a promising biodistribution profile with mainly signal in tumor (4.917 ± 0.776%ID/g, 2 h post-injection). Its rapid clearance from healthy tissues led to high tumor-to-organ ratios, resulting in highly contrasted PET images. These results were confirmed on subcutaneous xenografts of AsPC-1 tumor cells, a model of NTS1-positive human pancreatic adenocarcinoma.

[18F]FMISO PET/CT imaging of hypoxia as a non-invasive biomarker of disease progression and therapy efficacy in a preclinical model of pulmonary fibrosis: comparison with the [18F]FDG PET/CT approach.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor outcome and limited therapeutic options. Imaging of IPF is limited to high-resolution computed tomography (HRCT) which is often not sufficient for a definite diagnosis and has a limited impact on therapeutic decision and patient management. Hypoxia of the lung is a significant feature of IPF but its role on disease progression remains elusive. Thus, the aim of our study was to evaluate hypoxia imaging with [18F]FMISO as a predictive biomarker of disease progression and therapy efficacy in preclinical models of lung fibrosis in comparison with [18F]FDG. METHODS: Eight-week-old C57/BL6 mice received an intratracheal administration of bleomycin (BLM) at day (D) 0 to initiate lung fibrosis. Mice received pirfenidone (300 mg/kg) or nintedanib (60 mg/kg) by daily gavage from D9 to D23. Mice underwent successive PET/CT imaging at several stages of the disease (baseline, D8/D9, D15/D16, D22/D23) with [18F]FDG and [18F]FMISO. Histological determination of the lung expression of HIF-1α and GLUT-1 was performed at D23. RESULTS: We demonstrate that mean lung density on CT as well as [18F]FDG and [18F]FMISO uptakes are upregulated in established lung fibrosis (1.4-, 2.6- and 3.2-fold increase respectively). At early stages, lung areas with [18F]FMISO uptake are still appearing normal on CT scans and correspond to areas which will deteriorate towards fibrotic lesions at later timepoints. Nintedanib and pirfenidone dramatically and rapidly decreased mean lung density on CT as well as [18F]FDG and [18F]FMISO lung uptakes (pirfenidone: 1.2-, 2.9- and 2.6-fold decrease; nintedanib: 1.2-, 2.3- and 2.5-fold decrease respectively). Early [18F]FMISO lung uptake was correlated with aggressive disease progression and better nintedanib efficacy. CONCLUSION: [18F]FMISO PET imaging is a promising tool to early detect and monitor lung fibrosis progression and therapy efficacy.

Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.

Lipoproteins LDL versus HDL as nanocarriers to target either cancer cells or macrophages.

In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL, respectively) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of "Trojan horses" delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass spectometry and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anticancer immune response. Cumulative antitumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient blood can be used as natural nanocarriers allowing cell-specific targeting, paving the way toward more efficient, safer, and personalized use of chemotherapeutic and immunotherapeutic drugs in cancer.

Synthesis and evaluation of zirconium-89 labelled and long-lived GLP-1 receptor agonists for PET imaging.

INTRODUCTION: Lately, zirconium-89 has shown great promise as a radionuclide for PET applications of long circulating biomolecules. Here, the design and synthesis of protracted and long-lived GLP-1 receptor agonists conjugated to desferrioxamine and labelled with zirconium-89 is presented with the purpose of studying their in vivo distribution by PET imaging. The labelled conjugates were evaluated and compared to a non-labelled GLP-1 receptor agonist in both in vitro and in vivo assays to certify that the modification did not significantly alter the peptides' structure or function. Finally, the zirconium-89 labelled peptides were employed in PET imaging, providing visual verification of their in vivo biodistribution. METHODS: The evaluation of the radiolabelled peptides and comparison to their non-labelled parent peptide was performed by in vitro assays measuring binding and agonistic potency to the GLP-1 receptor, physicochemical studies aiming at elucidating change in peptide structure upon bioconjugation and labelling as well as an in vivo food in-take study illustrating the compounds' pharmacodynamic properties. The biodistribution of the labelled GLP-1 analogues was determined by ex vivo biodistribution and in vivo PET imaging. RESULTS: The results indicate that it is surprisingly feasible to design and synthesize a protracted, zirconium-89 labelled GLP-1 receptor agonist without losing in vitro potency or affinity as compared to a non-labelled parent peptide. Physicochemical properties as well as pharmacodynamic properties are also maintained. The biodistribution in rats shows high accumulation of radiolabelled peptide in well-perfused organs such as the liver, kidney, heart and lungs. The PET imaging study confirmed the findings from the biodistribution study with a significant high uptake in kidneys and presence of activity in liver, heart and larger blood vessels. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This initial study indicates the potential to monitor the in vivo distribution of long-circulating incretin hormones using zirconium-89 based PET.

Titanate Nanotubes Engineered with Gold Nanoparticles and Docetaxel to Enhance Radiotherapy on Xenografted Prostate Tumors.

Nanohybrids based on titanate nanotubes (TiONts) were developed to fight prostate cancer by intratumoral (IT) injection, and particular attention was paid to their step-by-step synthesis. TiONts were synthesized by a hydrothermal process. To develop the customengineered nanohybrids, the surface of TiONts was coated beforehand with a siloxane (APTES), and coupled with both dithiolated diethylenetriaminepentaacetic acidmodified gold nanoparticles (Au@DTDTPA NPs) and a heterobifunctional polymer (PEG3000) to significantly improve suspension stability and biocompatibility of TiONts for targeted biomedical applications. The prefunctionalized surface of this scaffold had reactive sites to graft therapeutic agents, such as docetaxel (DTX). This novel combination, aimed at retaining the AuNPs inside the tumor via TiONts, was able to enhance the radiation effect. Nanohybrids have been extensively characterized and were detectable by SPECT/CT imaging through grafted Au@DTDTPA NPs, radiolabeled with 111In. In vitro results showed that TiONtsAuNPsPEG3000DTX had a substantial cytotoxic activity on human PC3 prostate adenocarcinoma cells, unlike initial nanohybrids without DTX (Au@DTDTPA NPs and TiONtsAuNPsPEG3000). Biodistribution studies demonstrated that these novel nanocarriers, consisting of AuNP- and DTXgrafted TiONts, were retained within the tumor for at least 20 days on mice PC3 xenografted tumors after IT injection, delaying tumor growth upon irradiation.

Direct comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for 89Zr-immunoPET.

PURPOSE: Currently, the most commonly used chelator for labelling antibodies with 89Zr for immunoPET is desferrioxamine B (DFO). However, preclinical studies have shown that the limited in vivo stability of the 89Zr-DFO complex results in release of 89Zr, which accumulates in mineral bone. Here we report a novel chelator DFOcyclo*, a preorganized extended DFO derivative that enables octacoordination of the 89Zr radiometal. The aim was to compare the in vitro and in vivo stability of [89Zr]Zr-DFOcyclo*, [89Zr]Zr-DFO* and [89Zr]Zr-DFO. METHODS: The stability of 89Zr-labelled chelators alone and after conjugation to trastuzumab was evaluated in human plasma and PBS, and in the presence of excess EDTA or DFO. The immunoreactive fraction, IC50, and internalization rate of the conjugates were evaluated using HER2-expressing SKOV-3 cells. The in vivo distribution was investigated in mice with subcutaneous HER2+ SKOV-3 or HER2- MDA-MB-231 xenografts by PET/CT imaging and quantitative ex vivo tissue analyses 7 days after injection. RESULTS: 89Zr-labelled DFO, DFO* and DFOcyclo* were stable in human plasma for up to 7 days. In competition with EDTA, DFO* and DFOcyclo* showed higher stability than DFO. In competition with excess DFO, DFOcyclo*-trastuzumab was significantly more stable than the corresponding DFO and DFO* conjugates (p < 0.001). Cell binding and internalization were similar for the three conjugates. In in vivo studies, HER2+ SKOV-3 tumour-bearing mice showed significantly lower bone uptake (p < 0.001) 168 h after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (femur 1.5 ± 0.3%ID/g, knee 2.1 ± 0.4%ID/g) or [89Zr]Zr-DFO*-trastuzumab (femur 2.0 ± 0.3%ID/g, knee 2.68 ± 0.4%ID/g) than after injection with [89Zr]Zr-DFO-trastuzumab (femur 4.5 ± 0.6%ID/g, knee 7.8 ± 0.6%ID/g). Blood levels, tumour uptake and uptake in other organs were not significantly different at 168 h after injection. HER2- MDA-MB-231 tumour-bearing mice showed significantly lower tumour uptake (p < 0.001) after injection with [89Zr]Zr-DFOcyclo*-trastuzumab (16.2 ± 10.1%ID/g) and [89Zr]Zr-DFO-trastuzumab (19.6 ± 3.2%ID/g) than HER2+ SKOV-3 tumour-bearing mice (72.1 ± 14.6%ID/g and 93.1 ± 20.9%ID/g, respectively), while bone uptake was similar. CONCLUSION: 89Zr-labelled DFOcyclo* and DFOcyclo*-trastuzumab showed higher in vitro and in vivo stability than the current commonly used 89Zr-DFO-trastuzumab. DFOcyclo* is a promising candidate to become the new clinically used standard chelator for 89Zr immunoPET.

Modular Assembly of Multimodal Imaging Agents through an Inverse Electron Demand Diels-Alder Reaction.

The combination of two imaging probes on the same biomolecule gives access to targeted bimodal imaging agents that can provide more accurate diagnosis, complementary information, or that may be used in different applications, such as nuclear imaging and fluorescence guided surgery. In this study, we demonstrate that dichlorotetrazine, a small, commercially available compound, can be used as a modular platform to easily assemble various imaging probes. Doubly labeled tetrazines can then be conjugated to a protein through a biorthogonal IEDDA reaction. A series of difunctionalized tetrazine compounds containing various chelating agents and fluorescent dyes was synthesized. As a proof of concept, one of these bimodal probes was conjugated to trastuzumab, previously modified with a constrained alkyne group, and the resulting dual-labeled antibody was evaluated in a mouse model, bearing a HER2-positive tumor. A significant uptake into tumor tissues was observed in vivo, by both SPECT-CT and fluorescence imaging, and confirmed ex vivo in biodistribution studies.

Functionalization of theranostic AGuIX® nanoparticles for PET/MRI/optical imaging.

A novel trifunctional imaging probe containing a chelator of radiometal for PET, a NIR heptamethine cyanine dye, and a bioconjugatable handle, has been grafted onto AGuIX® nanoparticles via a Michael addition reaction. The resulting functionalized nanoparticles have been fully characterized, radiolabelled with 64Cu, and evaluated in a mice TSA tumor model using multimodal (PET/MRI/optical) imaging.

Functionalization of Gadolinium Chelates Silica Nanoparticle through Silane Chemistry for Simultaneous MRI/64Cu PET Imaging.

Multimodal nanoprobes are highly demanded for biomedical imaging applications to enhance the reliability of the diagnostic results. Among different types of nano-objects, ultrasmall silica gadolinium nanoparticle (SiGdNP) appears as a safe, effective, and versatile platform for this purpose. In this study, a new method to functionalize SiGdNP based on silane chemistry has been reported. Two types of chelating silanes (APTES-DOTAGA and APTES-NODAGA) have been synthesized and grafted on SiGdNP by a simple one-step protocol. This functionalization strategy requires no other reactants or catalyzers and does not compromise the ultrasmall size of the particles. NODAGA-functionalized particle has been labeled with 64Cu isotope and injected intravenously to mice bearing TS/A carcinoma tumor for biodistribution study to demonstrate its potential as a bimodal MRI/PET imaging agent. A fully integrated MRI/PET system was used to simultaneously monitor the distribution of the particle. The results showed that the functionalized particle maintained properties of a renal clearable NP which could rapidly escape through kidneys and had low retention in other organs, especially liver, even though its accumulation in the tumor was modest.

Site-specific near-infrared fluorescent labelling of proteins on cysteine residues with meso-chloro-substituted heptamethine cyanine dyes.

Near-infrared (NIR) fluorescence imaging is a promising new medical imaging modality. Associated with a targeting molecule, NIR fluorophores can accumulate selectively in tissues of interest and become valuable tools for the diagnosis and therapy of various pathologies. To facilitate the design of targeted NIR imaging agents, it is important to identify simple and affordable fluorescent probes, allowing rapid labelling of biovectors such as proteins, ideally in a site-specific manner. Here, we demonstrate that heptamethine cyanine based fluorophores, such as IR-783, that contain a chloro-cyclohexyl moiety within their polymethine chain can react selectively, at neutral pH, with cysteine residues in proteins to give stable, site-specifically labelled conjugates, that emit in the NIR spectral window. This reaction is exemplified with the labelling of peptides and two protein models: albumin and a Fab' antibody fragment. The resulting fluorescent proteins are stable and suitable for in vivo NIR imaging applications, as shown on a mice model. This straightforward one-step procedure, that does not require the prior derivatisation of the fluorophore with a bioconjugatable handle, should facilitate the production and use of near-infrared labelled proteins in life sciences.