CD206+ macrophages are relevant non-invasive imaging biomarkers and therapeutic targets in experimental lung fibrosis.

BACKGROUND: Interstitial lung diseases (ILDs) include a large number of diseases associated with progressive pulmonary fibrosis (PPF), including idiopathic pulmonary fibrosis (IPF). Despite the rarity of each of the fibrotic ILDs individually, they cumulatively affect a considerable number of patients. PPF is characterised by an excessive collagen deposition leading to functional decline. OBJECTIVES: Therapeutic options are limited to nintedanib and pirfenidone which are only able to reduce fibrosis progression. CD206-expressing M2 macrophages are involved in fibrosis progression, and whether they may be relevant therapeutic targets or biomarkers remains an open question. RESULTS: In our study, CD206+ lung macrophages were monitored in bleomycin-induced lung fibrosis in mice by combining flow cytometry, scRNAseq and in vivo molecular imaging using a single photon emission computed tomography (SPECT) radiopharmaceutical, 99mTc-tilmanocept. The antifibrotic effect of the inhibition of M2 macrophage polarisation with a JAK inhibitor, tofacitinib, was assessed in vivo. We demonstrate that CD206-targeted in vivo SPECT imaging with 99mTc-tilmanocept was able to accurately detect and quantify the increase in CD206+ macrophages from early to advanced stages of experimental fibrosis and ex vivo in lung biopsies from patients with IPF. CD206-targeted imaging also specifically detected a decrease in CD206+ lung macrophages on nintedanib and tofacitinib treatment. Importantly, early in vivo imaging of CD206+ macrophages allowed the prediction of experimental lung fibrosis progression as well as nintedanib and tofacitinib efficacy. CONCLUSIONS: These findings indicate that M2 macrophages may be relevant theranostic targets for personalised medicine for patients with PPF.

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.

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.

Oleanolic Acid Glycosides from Scabiosa caucasica and Scabiosa ochroleuca: Structural Analysis and Cytotoxicity.

In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-xylopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.

IL-1RAP, a Key Therapeutic Target in Cancer.

Cancer is a major cause of death worldwide and especially in high- and upper-middle-income countries. Despite recent progress in cancer therapies, such as chimeric antigen receptor T (CAR-T) cells or antibody-drug conjugate (ADC), new targets expressed by the tumor cells need to be identified in order to selectively drive these innovative therapies to tumors. In this context, IL-1RAP recently showed great potential to become one of these new targets for cancer therapy. IL-1RAP is highly involved in the inflammation process through the interleukins 1, 33, and 36 (IL-1, IL-33, IL-36) signaling pathways. Inflammation is now recognized as a hallmark of carcinogenesis, suggesting that IL-1RAP could play a role in cancer development and progression. Furthermore, IL-1RAP was found overexpressed on tumor cells from several hematological and solid cancers, thus confirming its potential involvement in carcinogenesis. This review will first describe the structure and genetics of IL-1RAP as well as its role in tumor development. Finally, a focus will be made on the therapies based on IL-1RAP targeting, which are now under preclinical or clinical development.

Development of Anti-LRRC15 Small Fragments for Imaging Purposes Using a Phage-Display ScFv Approach.

The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 107 cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10−3. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.

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.

[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.

Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence.

In this study, an original aza-BODIPY system comprising two Gd3+ complexes has been designed and synthesized for magnetic resonance imaging/optical imaging applications, by functionalization of the boron center. This strategy enabled the obtainment of a positively charged bimodal probe, which displays an increased water solubility, optimized photophysical properties in the near-infrared region, and very promising relaxometric properties. The absorption and emission wavelengths are 705 and 741 nm, respectively, with a quantum yield of around 10% in aqueous media. Moreover, the system does not produce singlet oxygen upon excitation, which would be toxic for tissues. The relaxivity obtained is high at intermediate fields (16.1 mM-1 s-1 at 20 MHz and 310 K) and competes with that of bigger or more rigid systems. A full relaxometric and 17O NMR study and fitting of the data using the Lipari-Szabo approach showed that this high relaxivity can be explained by the size of the system and the presence of some small aggregates. These optimized photophysical and relaxometric properties highlight the potential use of such systems for future bimodal imaging studies.

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.

A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging.

A new family of water-soluble and bioconjugatable aza-BODIPY fluorophores was designed and synthesized using a boron- functionalization strategy. These dissymmetric bis-ammonium aza-BODIPY dyes present optimal properties for a fluorescent probe; i.e., they are highly water-soluble, very stable in physiological medium; they do not aggregate in PBS, possess high quantum yield; and finally, they can be easily bioconjugated to antibodies. Preliminary in vitro and in vivo studies were performed for one of these fluorophores to image PD-L1 (Programmed Death-Ligand 1), highlighting the high potential of these new probes for future in vivo optical imaging studies.

Superparamagnetic Nanoparticle Delivery to the Cochlea Through Round Window by External Magnetic Field: Feasibility and Toxicity.

Introduction. The objective of this study was to evaluate the feasibility and toxicity of superparamagnetic iron oxide nanoparticles (SPIONs) administered into the cochlea through the round window (RW) by an external magnetic field. Materials and Methods. In 5 Wistar rats, the left RW was punctured. SPIONs suspended in hyaluronic gel (5 mg/mL) were applied in the RW niche and covered by a muscle graft. The nanoparticles were mobilized using a rare earth magnet (0.54 T) held in 4 consecutive positions around the head. The right ear served as control. Hearing function was monitored by auditory brainstem responses (4-32 kHz tone bursts). Results. The auditory thresholds remained unchanged 1 month after the administration. The histological study of the cochleae showed that SPIONs were driven into the scala tympani in the basal turn, the second turn, and the apex. Conclusion. Superparamagnetic nanoparticles can be driven inside the cochlea toward the apex with a preserved hearing up to 1 month in rats.

Near-Infrared-Emitting BODIPY-trisDOTA(111) In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations.

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl-BODIPY (BODIPY=boron-dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site. (111) In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY-chelators derivative as a bimodal imaging probe.

BODIPY: A Highly Versatile Platform for the Design of Bimodal Imaging Probes.

In molecular imaging, multimodal imaging agents can provide complementary information, for improving the accuracy of disease diagnosis or enhancing patient management. In particular, optical/nuclear imaging may find important preclinical and clinical applications. To simplify the preparation of dual-labeled imaging agents, we prepared versatile monomolecular multimodal imaging probe (MOMIP) platforms containing both a fluorescent dye (BODIPY) and a metal chelator (polyazamacrocycle). One of the MOMIP was conjugated to a cyclopeptide (i.e., octreotide) and radiolabeled with (111) In. In vitro and in vivo studies of the resulting bioconjugate were conducted, highlighting the potential of these BODIPY-based bimodal probes. This work also confirmed that the biovector and/or the bimodal probes must be chosen carefully, due to the impact of the MOMIP on the overall properties of the resulting imaging agent.

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.

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers.

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

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.

DOTAGA-trastuzumab. A new antibody conjugate targeting HER2/Neu antigen for diagnostic purposes.

Improved bifunctional chelating agents (BFC) are required for indium-111 radiolabeling of monoclonal antibodies (mAbs) under mild conditions to yield stable, target-specific agents. 2,2',2"-(10-(2,6-Dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-anhydride) was evaluated for mAb conjugation and labeling with indium-111. The DOTA analogue was synthesized and conjugated to trastuzumab-which targets the HER2/neu receptor-in mild conditions (PBS pH 7.4, 25 °C, 30 min) and gave a mean degree of conjugation of 2.6 macrocycle per antibody. Labeling of this immunoconjugate with indium-111 was performed in 75% yield after 1 h at 37 °C, and the proportion of (111)In-DOTAGA-trastuzumab reached 97% after purification. The affinity of DOTAGA-trastuzumab was 5.5 ± 0.6 nM as evaluated by in vitro saturation assays using HCC1954 breast cancer cell line. SPECT/CT imaging and biodistribution studies were performed in mice bearing breast cancer BT-474 xenografts. BT-474 tumors were clearly visualized on SPECT images at 24, 48, and 72 h postinjection. The tumor uptake of [(111)In-DOTAGA]-trastuzumab reached 65%ID/g 72 h postinjection. These results show that the DOTAGA BFC appears to be a valuable tool for biologics conjugation.

Respiratory gated PET/CT in a European multicentre retrospective study: added diagnostic value in detection and characterization of lung lesions.

PURPOSE: The aim of our work is to evaluate the added diagnostic value of respiratory gated (4-D) positron emission tomography/computed tomography (PET/CT) in lung lesion detection/characterization in a large patient population of a multicentre retrospective study. METHODS: The data of 155 patients (89 men, 66 women, mean age 63.9 ± 11.1 years) from 5 European centres and submitted to standard (3-D) and 4-D PET/CT were retrospectively analysed. Overall, 206 lung lesions were considered for the analysis (mean ± SD lesions dimension 14.7 ± 11.8 mm). Maximum standardized uptake values (SUV(max)) and lesion detectability were assessed for both 3-D and 4-D PET/CT studies; 3-D and 4-D PET/CT findings were compared to clinical follow-up as standard reference. RESULTS: Mean ± SD 3-D and 4-D SUV(max) values were 5.2 ± 5.1 and 6.8 ± 6.1 (p < 0.0001), respectively, with an average percentage increase of 30.8 %. In 3-D PET/CT, 86 of 206 (41.7 %) lesions were considered positive, 70 of 206 (34 %) negative and 50 of 206 (24.3 %) equivocal, while in 4-D PET/CT 117 of 206 (56.8 %) lesions were defined as positive, 80 of 206 (38.8 %) negative and 9 of 206 (4.4 %) equivocal. In 34 of 50 (68 %) 3-D equivocal lesions follow-up data were available and the presence of malignancy was confirmed in 21 of 34 (61.8 %) lesions, while in 13 of 34 (38.2 %) was excluded. In 31 of these 34 controlled lesions, 20 of 34 (58.8 %) and 11 of 34 (32.4 %) were correctly classified by 4-D PET/CT as positive and negative, respectively; 3 of 34 (8.8 %) remained equivocal. With equivocal lesions classified as positive, the overall accuracy of 3-D and 4-D was 85.7 and 92.8 %, respectively, while the same figures were 80.5 and 94.2 % when equivocal lesions were classified as negative. CONCLUSION: The respiratory gated PET/CT technique is a valuable clinical tool in diagnosing lung lesions, improving quantification and confidence in reporting, reducing 3-D undetermined findings and increasing the overall accuracy in lung lesion detection and characterization.