Gadolinium-Based Nanoparticles Sensitize Ovarian Peritoneal Carcinomatosis to Targeted Radionuclide Therapy.

Ovarian cancer (OC) is the most lethal gynecologic malignancy (5-y overall survival rate, 46%). OC is generally detected when it has already spread to the peritoneal cavity (peritoneal carcinomatosis). This study investigated whether gadolinium-based nanoparticles (Gd-NPs) increase the efficacy of targeted radionuclide therapy using [177Lu]Lu-DOTA-trastuzumab (an antibody against human epidermal growth factor receptor 2). Gd-NPs have radiosensitizing effects in conventional external-beam radiotherapy and have been tested in clinical phase II trials. Methods: First, the optimal activity of [177Lu]Lu-DOTA-trastuzumab (10, 5, or 2.5 MBq) combined or not with 10 mg of Gd-NPs (single injection) was investigated in athymic mice bearing intraperitoneal OC cell (human epidermal growth factor receptor 2-positive) tumor xenografts. Next, the therapeutic efficacy and toxicity of 5 MBq of [177Lu]Lu-DOTA-trastuzumab with Gd-NPs (3 administration regimens) were evaluated. NaCl, trastuzumab plus Gd-NPs, and [177Lu]Lu-DOTA-trastuzumab alone were used as controls. Biodistribution and dosimetry were determined, and Monte Carlo simulation of energy deposits was performed. Lastly, Gd-NPs' subcellular localization and uptake, and the cytotoxic effects of the combination, were investigated in 3 cancer cell lines to obtain insights into the involved mechanisms. Results: The optimal [177Lu]Lu-DOTA-trastuzumab activity when combined with Gd-NPs was 5 MBq. Moreover, compared with [177Lu]Lu-DOTA-trastuzumab alone, the strongest therapeutic efficacy (tumor mass reduction) was obtained with 2 injections of 5 mg of Gd-NPs/d (separated by 6 h) at 24 and 72 h after injection of 5 MBq of [177Lu]Lu-DOTA-trastuzumab. In vitro experiments showed that Gd-NPs colocalized with lysosomes and that their radiosensitizing effect was mediated by oxidative stress and inhibited by deferiprone, an iron chelator. Exposure of Gd-NPs to 177Lu increased the Auger electron yield but not the absorbed dose. Conclusion: Targeted radionuclide therapy can be combined with Gd-NPs to increase the therapeutic effect and reduce the injected activities. As Gd-NPs are already used in the clinic, this combination could be a new therapeutic approach for patients with ovarian peritoneal carcinomatosis.

Fc-engineered monoclonal antibodies to reduce off-target liver uptake.

BACKGROUND: Radiolabeled-antibodies usually display non-specific liver accumulation that may impair image analysis and antibody biodistribution. Here, we investigated whether Fc silencing influenced antibody biodistribution. We compared recombinant 89Zr-labeled antibodies (human IgG1 against different targets) with wild-type Fc and with mutated Fc (LALAPG triple mutation to prevent binding to Fc gamma receptors; FcγR). After antibody injection in mice harboring xenografts of different tumor cell lines or of immortalized human myoblasts, we analyzed antibody biodistribution by PET-CT and conventional biodistribution analysis. RESULTS: Accumulation in liver was strongly reduced and tumor-specific targeting was increased for the antibodies with mutated Fc compared with wild-type Fc. CONCLUSION: Antibodies with reduced binding to FcγR display lower liver accumulation and better tumor-to-liver ratios. These findings need to be taken into account to improve antibody-based theragnostic approaches.

In vivo validation of 68Ga-labeled AMD3100 conjugates for PET imaging of CXCR4.

INTRODUCTION: The chemokine receptor CXCR4 has been shown to be over-expressed in multiple types of cancer and is usually associated with aggressive phenotypes and poor prognosis. Successfully targeting and imaging the expression level of this receptor in tumours could inform treatment selection and facilitate patient stratification. METHODS: Known conjugates of AMD3100 that are specific to CXCR4 have been radiolabelled with gallium-68 and evaluated in naïve and tumour-bearing mice. Tumour uptake of the radiotracers was compared to the known CXCR4-specific PET imaging agent, [68Ga]Pentixafor. RESULTS: Ex vivo biodistribution in naïve animals showed CXCR4-mediated uptake in the liver with both radiotracers, confirmed by blocking experiments with the high affinity CXCR4 antagonist Cu2CB-Bicyclam (IC50 = 3 nM). PET/CT imaging studies revealed one tracer to have a higher accumulation in the tumour (SUVMean of 0.89 ± 0.14 vs 0.32 ± 0.11). CXCR4-specificity of the best performing tracer was confirmed by administration of a blocking dose of Cu2CB-Bicyclam, showing a 3- and 6-fold decrease in tumour and liver uptake, respectively. CONCLUSION AND ADVANCES IN KNOWLEDGE: This initial study offers some interesting insights on the impact of some structural features on the pharmacokinetics and metabolic stability of the radiotracer. Additionally, as Pentixafor only binds to human CXCR4, the development of CXCR4-targeted imaging agents that bind to the receptor across different species could significantly help with preclinical evaluation of new CXCR4-specific therapeutics.

Identification of new FK866 analogues with potent anticancer activity against pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases for which chemotherapy has not been very successful yet. FK866 ((E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) is a well-known NAMPT (nicotinamide phosphoribosyltransferase) inhibitor with anti-cancer activities, but it failed in phase II clinical trials. We found that FK866 shows anti-proliferative activity in three PDAC cell lines, as well as in Jurkat T-cell leukemia cells. More than 50 FK866 analogues were synthesized that introduce substituents on the phenyl ring of the piperidine benzamide group of FK866 and exchange its buta-1,4-diyl tether for 1-oxyprop-3-yl, (E)-but-2-en-1,4-diyl and 2- and 3-carbon tethers. The pyridin-3-yl moiety of FK866 was exchanged for chlorinated and fluorinated analogues and for pyrazin-2-yl and pyridazin-4-yl groups. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity. Our best cell anti-proliferative compounds were the 2,4,6-trimethoxybenzamide analogue of FK866 ((E)-N-(4-(1-(2,4,6-trimethoxybenzoyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) (9), the 2,6-dimethoxybenzamide (8) and 2-methoxybenzamide (4), which exhibited an IC50 of 0.16 nM, 0.004 nM and 0.08 nM toward PDAC cells, respectively.

From the target cell theory to a more integrated view of radiobiology in Targeted radionuclide therapy: The Montpellier group's experience.

Targeted radionuclide therapy (TRT) is used to treat disseminated or metastatic tumours in which conventional external beam radiotherapy (EBRT) would have unacceptable side effects. Unlike EBRT, TRT delivers low doses at a continuous low dose rate. In EBRT, the effect increases progressively with the dose rate, and biological effects (tumour control and normal tissue damage) are related to the dose according to a sigmoid curve model. This model is part of the so-called quantitative radiobiology that is mostly based on the target cell theory, according to which cell death is due to (lethal) radiation hits to vital cellular targets. This model was developed for EBRT, but was adapted to low dose-rate situations by including a parameter that reflects the time needed to repair tissue damage. However, a growing body of evidence indicates that the model should take into account also the biological effects, which are due to intercellular communications (bystander effects) and amplify the effects of radiation, as well as the immune system. Moreover, extranuclear targets must be considered, although induced intracellular and intercellular signalling pathways may ultimately result in DNA damage. It is likely that bystander effects and immune response always contribute to the overall response to TRT at different levels, and that dose and dose rate are key parameters in controlling their real contribution. We hypothesize that the dose rate is the key determinant in the balance between the physical and DNA-centred response on one side, and the biological response that integrates all subcellular compartments and intercellular signalling pathways on the other side.

Therapeutic antibodies - natural and pathological barriers and strategies to overcome them.

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.

Targeted Radionuclide Therapy Using Auger Electron Emitters: The Quest for the Right Vector and the Right Radionuclide.

Auger electron emitters (AEEs) are attractive tools in targeted radionuclide therapy to specifically irradiate tumour cells while sparing healthy tissues. However, because of their short range, AEEs need to be brought close to sensitive targets, particularly nuclear DNA, and to a lower extent, cell membrane. Therefore, radioimmunoconjugates (RIC) have been developed for specific tumour cell targeting and transportation to the nucleus. Herein, we assessed, in A-431CEA-luc and SK-OV-31B9 cancer cells that express low and high levels of HER2 receptors, two 111In-RIC consisting of the anti-HER2 antibody trastuzumab conjugated to NLS or TAT peptides for nuclear delivery. We found that NLS and TAT peptides improved the nuclear uptake of 111In-trastuzumab conjugates, but this effect was limited and non-specific. Moreover, it did not result in a drastic decrease of clonogenic survival. Indium-111 also contributed to non-specific cytotoxicity in vitro due to conversion electrons (30% of the cell killing). Comparison with [125I]I-UdR showed that the energy released in the cell nucleus by increasing the RIC's nuclear uptake or by choosing an AEE that releases more energy per decay should be 5 to 10 times higher to observe a significant therapeutic effect. Therefore, new Auger-based radiopharmaceuticals need to be developed.

89Zr-PET imaging of DNA double-strand breaks for the early monitoring of response following α- and ß-particle radioimmunotherapy in a mouse model of pancreatic ductal adenocarcinoma.

Rationale: The evaluation of early treatment response is critical for patient prognosis and treatment planning. When the current methods rely on invasive protocols that evaluate the expression of DNA damage markers on patient biopsy samples, we aim to evaluate a non-invasive PET imaging approach to monitor the early expression of the phosphorylated histone γH2AX in the context of pancreatic cancer targeted radionuclide therapy. Pancreatic ductal adenocarcinoma has a poor patient prognosis due to the absence of curative treatment for patients with advanced disease. There is therefore a critical need for the fast clinical translation of new therapeutic options. In line with these observations, our group has been focusing on the development of radiotheranostic agents based on a fully human monoclonal antibody (5B1) with exceptional affinity for CA19.9, an antigen overexpressed in PDAC. Two on-going clinical trials resulted from these efforts, one with 89Zr (diagnosis) and one with 177Lu (ß-particle therapy). More recently, we successfully developed and evaluated in PDAC mouse models a targeted α-therapy strategy with high clinical translation potential. We aim to expedite the clinical translation of the developed radioimmunotherapy approaches by investigating the early therapeutic response and effect of radiation therapy in a PDAC mouse model via PET imaging. Methods: Mice bearing BxPC3 tumor xenografts were treated with α- and ß-particle pretargeted radioimmunotherapy (PRIT), external beam radiotherapy (EBRT), or sham-treated (vehicle). The phosphorylated histone γH2AX produced as a response to DNA double strand breaks was quantified with the PET radiotracer, [89Zr]Zr-DFO-anti-γH2AX-TAT. Results: PET imaging studies in BxPC3 PDAC mouse models demonstrated increased uptake of [89Zr]Zr-DFO-anti-γH2AX-TAT (6.29 ± 0.15 %IA/g) following ß-PRIT in BxPC3 PDAC xenografts as compared to the saline control group (4.58 ± 0.76 %IA/g) and EBRT control group (5.93 ± 0.76 %IA/g). Similarly, significantly higher uptake of [89Zr]Zr-DFO-anti-γH2AX-TAT was observed in tumors of the 225Ac-PRIT and EBRT (10 Gy) cohorts (7.37 ± 1.23 and 6.80 ± 1.24 %IA/g, respectively) compared to the negative control cohort (5.08 ± 0.95 %IA/g). Ex vivo γH2AX immunohistochemistry and immunofluorescence analysis correlated with in vivo89Zr-anti-γH2AX PET/CT imaging with increased γH2AX positive cell and γH2AX foci per cell in the treated cohorts. When α-PRIT resulted in prolonged overall survival of treated animals (107.5 days) as compared to ß-PRIT (73.0 days), no evidence of difference in [89Zr]Zr-DFO-anti-γH2AX-TAT uptake at the tumor site was observed, highlighting that DNA damage is not the sole radiobiology paradigm and that off-targeted (bystander) effects should be considered. Conclusions: PET imaging studies with [89Zr]Zr-DFO-anti-γH2AX-TAT following α- and ß-particle PRIT in a BxPC3 PDAC subcutaneous xenograft mouse model allowed the monitoring of tumor radiobiological response to treatment.

Innovative Magnetic Nanoparticles for PET/MRI Bimodal Imaging.

Superparamagnetic iron oxide nanoparticles were developed as positron emission tomography (PET) and magnetic resonance imaging (MRI) bimodal imaging agents. These nanoparticles (NPs), with a specific nanoflower morphology, were first synthesized and simultaneously functionalized with 3,4-dihydroxy-l-phenylalanine (LDOPA) under continuous hydrothermal conditions. The resulting NPs exhibited a low hydrodynamic size of 90 ± 2 nm. The functional groups of LDOPA (-NH2 and -COOH) were successfully used for the grafting of molecules of interest in a second step. The nanostructures were modified by poly(ethylene glycol) (PEG) and a new macrocyclic chelator MANOTA for further 64Cu radiolabeling for PET imaging. The functionalized NPs showed promising bimodal (PET and MRI) imaging capability with high r 2 and r 2* (T 2 and T 2* relaxivities) values and good stability. They were mainly uptaken from liver and kidneys. No cytotoxicity effect was observed. These NPs appear as a good candidate for bimodal tracers in PET/MRI.

A rapid bead-based radioligand binding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals.

INTRODUCTION: Determination of the target-binding fraction (TBF) of radiopharmaceuticals using cell-based assays is prone to inconsistencies arising from several intrinsic and extrinsic factors. Here, we report a cell-free quantitative method of analysis to determine the TBF of radioligands. METHODS: Magnetic beads functionalized with Ni-NTA or streptavidin were incubated with 1 µg of histidine-tagged or biotinylated antigen of choice for 15 min, followed by incubating 1 ng of the radioligand for 30 min. The beads, supernatant and wash fractions were measured for radioactivity on a gamma counter. The TBF was determined by quantifying the percentage of activity associated with the magnetic beads. RESULTS: The described method works robustly with a variety of radioisotopes and class of molecules used as radioligands. The entire assay can be completed within 2 h. CONCLUSION: The described method yields results in a rapid and reliable manner whilst improving and extending the scope of previously described bead-based radioimmunoassays. ADVANCES IN KNOWLEDGE: Using a bead-based radioligand binding assay overcomes the limitations of traditional cell-based assays. The described method is applicable to antibody as well as non-antibody based radioligands and is independent of the effect of target antigen density on cells, the choice of radioisotope used for synthesis of the radioligand and the temperature at which the assay is performed. IMPLICATIONS FOR PATIENT CARE: The bead-based radioligand binding assay is significantly easier to perform and is ideally suited for adoption by the radiopharmacy as a quality control method of analysis to fulfill the criteria for release of radiopharmaceuticals in the clinic. The use of this assay is likely to ensure a more reliable validation of radiopharmaceutical quality and result in fewer failed doses, which could ultimately translate to an efficient release of radiopharmaceuticals for administration to patients in the clinic.

Trastuzumab gold-conjugates: synthetic approach and in vitro evaluation of anticancer activities in breast cancer cell lines.

We describe the preparation of gold(i)-compounds that are amenable to efficient bioconjugation with monoclonal antibodies via activated ester or maleimide linkers. New Trastuzumab-gold conjugates were synthesized and fully characterized. These bioconjugates are significantly more cytotoxic (sub-micromolar range) to HER2-positive breast cancer cells than the gold complexes and Trastuzumab.

Leveraging Bioorthogonal Click Chemistry to Improve 225Ac-Radioimmunotherapy of Pancreatic Ductal Adenocarcinoma.

PURPOSE: Interest in targeted alpha-therapy has surged due to α-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels-Alder ligation between an 225Ac-labeled tetrazine radioligand and a trans-cyclooctene-bearing anti-CA19.9 antibody (5B1) for pretargeted α-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT).Experimental Design: A side-by-side comparison of 225Ac-PRIT and conventional RIT using a directly 225Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models. RESULTS: A comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225Ac-radioimmunoconjugate but-importantly-further allowed for the ex vivo monitoring of 225Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness. CONCLUSIONS: The ability of 225Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.

α-Emitters for Radiotherapy: From Basic Radiochemistry to Clinical Studies-Part 2.

The use of radioactive sources to deliver cytotoxic ionizing radiation to disease sites dates back to the early 20th century, with the discovery of radium and its physiologic effects. α-emitters are of particular interest in the field of clinical oncology for radiotherapy applications. The first part of this review explored the basic radiochemistry, high cell-killing potency, and availability of α-emitting radionuclides, together with hurdles such as radiolabeling methods and daughter redistribution. The second part of this review will give an overview of the most promising and current uses of α-emitters in preclinical and clinical studies.

α-Emitters for Radiotherapy: From Basic Radiochemistry to Clinical Studies-Part 1.

With a short particle range and high linear energy transfer, α-emitting radionuclides demonstrate high cell-killing efficiencies. Even with the existence of numerous radionuclides that decay by α-particle emission, only a few of these can reasonably be exploited for therapeutic purposes. Factors including radioisotope availability and physical characteristics (e.g., half-life) can limit their widespread dissemination. The first part of this review will explore the diversity, basic radiochemistry, restrictions, and hurdles of α-emitters.

Preclinical optimization of antibody-based radiopharmaceuticals for cancer imaging and radionuclide therapy-Model, vector, and radionuclide selection.

Intact antibodies and their truncated counterparts (eg, Fab, scFv fragments) are generally exquisitely specific and selective vectors, enabling recognition of individual cancer-associated molecular phenotypes against a complex and dynamic biomolecular background. Complementary alignment of these advantages with unique properties of radionuclides is a defining paradigm in both radioimmunoimaging and radioimmunotherapy, which remain some of the most adept and promising tools for cancer diagnosis and treatment. This review discusses how translational potency can be maximized through rational selection of antibody-nuclide couples for radioimmunoimaging/therapy in preclinical models.

AMD3100: A Versatile Platform for CXCR4 Targeting (68)Ga-Based Radiopharmaceuticals.

CXCR4 is a G protein-coupled receptor (GPCR), which is overexpressed in numerous diseases, particularly in multiple cancers. Therefore, this receptor represents a valuable target for imaging and therapeutic purposes. Among the different approaches, which were developed for CXCR4 imaging, a CXCR4 antagonist biscyclam system (AMD3100, also called Mozobil), currently used in the clinic for the mobilization of hematopoietic stem cells, was radiolabeled with different radiometals such as (62)Zn, (64)Cu, (67)Ga, or (99m)Tc. However, cyclam is not an ideal chelator for most of these radiometals, and could lead to the release of the radionuclide in vivo. In the current study, a new family of CXCR4 imaging agents is presented, in which AMD3100 is used as a carrier for specific delivery of an imaging reporter, i.e., a (68)Ga complex for PET imaging. AMD3100 was functionalized on the phenyl moiety with different linkers, either ethylenediamine or diamino-polyethylene glycol 3 (PEG3). The resulting AMD3100 analogues were further coupled with two different chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). Five potential CXCR4 targeting agents were obtained. The derived AMD3100-based ligands were labeled with (68)Ga, highlighting the influence of the spacer nature on the (68)Ga-labeling yield. The lipophilic character of the different systems was also investigated, as well as their affinity for the CXCR4 receptor. The most promising compound was further evaluated in vivo in H69 tumor xenografts by biodistribution and PET imaging studies, validating the proof of principle of our concept.

MA-NOTMP: A Triazacyclononane Trimethylphosphinate Based Bifunctional Chelator for Gallium Radiolabelling of Biomolecules.

In the past few years, gallium-68 has demonstrated significant potential as a radioisotope for positron emission tomography (PET), and the optimization of chelators for gallium coordination is a major goal in the development of radiopharmaceuticals. Methylaminotriazacyclononane trimethylphosphinate (MA-NOTMP), a new C-functionalized triazacyclononane derivative with phosphinate pendant arms, presents excellent coordination properties for (68) Ga (low ligand concentration, labelling at low pH even at room temperature). A "ready-to-be-grafted" bifunctional chelating agent (p-NCS-Bz-MA-NOTMP) was prepared to allow (68) Ga labelling of sensitive biological vectors. Conjugation to a bombesin(7-14) derivative was performed, and preliminary in vitro experiments demonstrated the potential of MA-NOTMP in the development of radiopharmaceuticals. This new chelator is therefore of major interest for labelling sensitive biomolecules, and further in vivo experiments will soon be performed.

New AMD3100 derivatives for CXCR4 chemokine receptor targeted molecular imaging studies: synthesis, anti-HIV-1 evaluation and binding affinities.

CXCR4 is a target of growing interest for the development of new therapeutic drugs and imaging agents as its role in multiple disease states has been demonstrated. AMD3100, a CXCR4 chemokine receptor antagonist that is in current clinical use as a haematopoietic stem cell mobilising drug, has been widely studied for its anti-HIV properties, potential to inhibit metastatic spread of certain cancers and, more recently, its ability to chelate radiometals for nuclear imaging. In this study, AMD3100 is functionalised on the phenyl moiety to investigate the influence of the structural modification on the anti-HIV-1 properties and receptor affinity in competition with anti-CXCR4 monoclonal antibodies and the natural ligand for CXCR4, CXCL12. The effect of complexation of nickel(II) in the cyclam cavities has been investigated. Two amino derivatives were obtained and are suitable intermediates for conjugation reactions to obtain CXCR4 molecular imaging agents. A fluorescent probe (BODIPY) and a precursor for (18)F (positron emitting isotope) radiolabelling were conjugated to validate this route to new CXCR4 imaging agents.