PRISMAP funded projects

Currently, there is no one technique that combines all the desirable properties for imaging e.g. high sensitivity/resolution, low cost and general availability, but multimodality imaging, merging two or more imaging techniques, has the potential to overcome this. In particular, positron emission tomography (PET) with magnetic resonance imaging (MRI) combines the superior sensitivity of PET with the high spatial resolution and soft tissue contrast of MRI and is gaining traction as a key bridge for precise radiotherapy. Despite the recognition that multimodality imaging has several benefits, there have been limited advances in the development of suitable imaging tools in clinical use. We have proposed new multimodal imaging agents that combine 152-Tb for PET with Gd for MRI. Through PRISMAP, we seek to optimise the radiolabelling conditions of our new imaging agents, confirm stability, and suitability for PET/MRI. This proof-of-concept study will allow us to further optimise these imaging agents for the next generation of PET/MRI probe.

With the revolution of targeted therapy, theranostics and novel applications in Positron Emission Tomography (PET), new radionuclides for PET imaging have gained attention as possible candidates for clinical applications. Manganese-52 in its ground state (52Mn) presents interesting characteristics, including a relatively long half-life (5.6 days), suitable for long studies, and the emission of a cascade of multiple prompt γ rays within the β+ decay. Although often the emission of such gamma rays is regarded as a limitation in PET imaging, introducing a background of spurious coincidences, novel applications such as the study of Positronium Lifetime Imaging (PLI) or multiplexed PET (mPET) are based on the detection of the multiple gamma coincidences triggered by the emission of prompt gamma rays with the positron. Moreover, the average energy of the emitted positrons from 52Mn is low ( 242 keV average energy and 575 keV maximum energy) compared with other non-standard isotopes for PET such as 124I, 44Sc or 68Ga, exhibiting a positron range even more favorable than the one of the widely used 18F. 52Mn has been proposed for clinical applications as a labeling agent for immunotherapy labeling DOTA chelates; for theranostics, monitoring HER2+ tumors in breast cancer; and for applications in neuroimaging to trace neuronal connections. These features make 52Mn an excellent candidate for applications in PET involving multiple coincidences detection (e.g. new applications and broadening the current applicability for PLI and mPET) and for furthering their clinical translation. This project is a collaboration between Jagiellonian University (Poland), Inselspital Bern (Switzerland) and Complutense University of Madrid (Spain). Application of 52Mn for PLI and mPET will be studied in J-PET scanner (Krakow), SEDECAL SuperArgus pre-clinical scanner (Madrid) and Siemens Biograph Vision Quadra (Bern).

Currently, nuclear medicine is witnessing a growing interest in theranostic strategies that integrate diagnostics and therapy into a single tool. Innovative solutions involving nanomaterials are being sought for their unique properties that can significantly enhance treatment effectiveness and diagnostic precision. The scientific goal of this project is to develop and synthesize novel nano-radiopharmaceutical tools for theranostic applications, combining diagnostic and therapeutic capabilities in one platform. These tools will utilize a new generation of luminescent up-converting nanoparticles (UCNPs), which will be radiolabelled with therapeutic radionuclides for both imaging and treatment. UCNPs are inorganic nanomaterials composed of matrices like fluorides, oxides, or sulphides, doped with lanthanide ions. Their distinct spectroscopic properties allow them to emit light in the visible (VIS) or ultraviolet (UV) spectrum upon excitation with near-infrared radiation (NIR), a process known as NIR-to-VIS/UV upconversion, offering advantages in biomedical applications such as reduced background fluorescence and deeper tissue penetration. The project focuses on the radiomodification of UCNPs using the beta-emitting radionuclides ytterbium-175 (¹⁷⁵Yb) and erbium-169 (¹⁶⁹Er). ¹⁷⁵Yb emits beta radiation effective for targeted cancer therapy and releases gamma photons suitable for SPECT imaging. Meanwhile, ¹⁶⁹Er provides therapeutic benefits with minimal gamma emission, reducing radiation exposure to healthy tissues while delivering effective beta radiation to tumors. These radionuclides are ideal for precise, targeted cancer therapies, facilitating personalized and less invasive medical interventions. This project represents a pioneering effort to create a novel theranostic platform by radiolabelling UCNPs with ¹⁷⁵Yb and ¹⁶⁹Er. To our knowledge, this is the first global attempt to apply these radionuclides in a theranostic context, highlighting the innovative potential.

Disulphide rebridging is a site-specific bioconjugation of prosthetic groups to biomolecules. Most therapeutically relevant biomolecules contain at least one disulphide bridge available for the construction of disulphide rebridge. This strategy proved to be an effective for the functionalization of peptides, monoclonal antibodies, and their fragments. However, the potential of rebridging strategy has received relatively little attention in the context of radiolabelling. During the course of the project, we would like to explore the potential of this technique in application to radiotherapy. Currently, our group is researching the application of this technique to therapy with iodine-131, during this project we would like to test the application to targeted alpha therapy. Astatine-211 is an α-emitting radionuclide and is currently of major interest for the development of therapeutic radiopharmaceuticals since α-therapy is gaining tremendous attention nowadays. Astatine-211 has a convenient half-life of 7.21 h, which is long enough to perform an effective treatment. The specific goal of this project is to develop new prosthetic groups for astatination of biomolecules via disulphide rebridging. We have prepared prosthetic groups with suitable leaving groups allowing radiohalogen labelling to be carried out. In addition, we would like to test heterocyclic-based prosthetic groups, the structure of which should help to stabilise the carbon-astatine bond to reduce the risk of deastatination in vivo. Initial labelling studies, conjugation with octreotide and in vitro testing of the compounds produced will first be carried out with iodine-131 to develop the best reaction parameters and select the most promising prosthetic groups for further testing. Once positive results are obtained, we will carry out labelling with astatine-211 and the resulting astatinated prosthetic groups conjugated with octreotide via disulphide rebridging will be tested in primary in vitro studies.

In this proposal, we will employ a multidisciplinary cooperation, bringing state-of-the art metrology to the preclinical and clinical practice within the framework of the ongoing European research project 22HLT03 AlphaMet. Requested support from the PRISMAP consortium will help to meet some of the AlphaMet's most important goals: comparison and calibration of radionuclide activity calibrators at clinical and preclinical centers in several European countries and multicenter comparison in quantitative SPECT imaging of alpha emitters. Effective implementation of dosimetry-guided targeted alpha therapy (TAT) as mandated by the basic safety standards (BSS) requires end-to-end traceability to primary standards: from accurate measurements of the activity administered to patients, to the calculation of the absorbed doses, with robust uncertainty budgets Given that the use of alpha emitters is still in its early clinical stages as compared to established therapies using beta emitters, there is an opportunity for this project to come in earlier in the path towards routine clinical implementation, and to provide the required measurement capabilities with known uncertainties that will contribute to support robust measurement capabilities that improve confidence in the use of personalized TAT.

Scandium radioisotopes have attracted significant attention for their potential use in oncology. The applicability of Sc-44 for positron emission tomography (PET) is clinically proved and preclinically studies emphasized the use of Sc-47 in targeted β- therapy. These radionuclides can be used for labelling a molecular target of high therapeutic interest and sequentially used for diagnosis and treatment. This approach is called theranostics and requires a suitable bifunctional chelator (BFC) for incorporation of the radionuclide and targeting. DOTA, the single clinical tested chelator for scandium radioisotopes, and recently explored cyclic chelators require harsh labelling conditions. This impedes labelling of sensitive targeting moieties (e.g. single-chain fragment variable, scFv) of high clinical interest. Acyclic chelators with better labelling kinetics however showed release of the radionuclide. The aim of this project is therefore to develop novel scandium-specific BFCs that can be radiolabelled under physiological condition while maintaining a high kinetic inertness. For that, a library of chelators will be explored for radiolabelling with Sc-47 under various conditions. Chelators with high labelling yields will be studied for stabilities against biological competitors, which allows to select those for conjugation to a prostate-specific membrane antigen (PSMA) targeting unit. The conjugates will be assessed for their labelling behavior with Sc-47 and successfully labelled radioconjugates will then be investigated on prostate cancer cell lines (LnCAP) to assess their in vitro stability and specify for tumour cells. Finally, lead theranostics will be studied in vivo to show their imaging and therapeutic efficacy in murine models of prostate cancer. The outcome of this project will be a defined radiolabelling methodology for a bifunctional chelator with Sc-47 in an in vitro and in vivo proof-of-principle study of scandium radionuclides in theranostics.

Despite recent progress, an unmet clinical need remains for novel treatments to improve cancer outcomes. Targeted radiotheranostics – radiopharmaceuticals coupled with diagnostic and therapeutic radionuclides - are highly promising to improve therapeutic efficacy of anticancer therapy with less toxicity. Within this field, fibroblast activation protein (FAP)-targeted radiotheranostics have been subject of intense investigation. FAP is a serine protease highly expressed in cancer-associated fibroblasts (CAFs). These CAFs are a group of tumor stromal cells typically associated with cancer growth and metastasis. Despite tremendous progress on FAP ligands for tumor diagnosis, an unmet clinical need remains for radionuclide therapies targeting FAP. One of the reasons is their short tumor residence time. Therefore, in this application we aim to develop novel FAP-targeting radiotheranostics with improved tumor retention, while limiting the overall background activity.

Neuroendocrine tumours (NETs) can originate in almost every organ, but most commonly arise in the GI tract, lung and pancreas. The heterogeneity of NETs, varying in primary site, histology and aggressiveness, leads to difficulties in treatment development. Targeted radionuclide therapies (TRT) have been driven by beta particle-emitters such as Lutetium-177 (177Lu), with the most used NET TRT being 177Lu-DOTATATE. Investigation into the therapeutic potential of terbium-161 (161Tb) is of particular interest due to its unique decay characteristics. The addition of Auger electron emissions could make 161Tb a more effective cancer treatment than 177Lu. Due to their similarities in coordination chemistry, 161Tb can often be directly substituted for 177Lu. Of note,161Tb has successfully been coordinated with DOTA. However, the high temperatures and reaction times required for DOTA complexation limit its biological research and therefore its future clinical potential. As a result, we have begun preliminary studies of novel chelators which we would like to further develop with 161Tb. A restriction to the study of TRT in cancer treatment is a lack of suitable models. This limitation is particularly prominent for NETs where their diverse origins do not allow one model to be representative. Whilst 2D cell line propagation has underpinned in vitro cancer research for decades, 3D models better depict tumour structure and microenvironment. Therefore, we have begun to explore a range of 2D NET cell lines that can be used to generate spheroids. This will allow for direct comparisons between 2D and 3D models, leading to the generation of novel tools for in the evaluation of TRT in NETs. We will carry out initial in vitro studies on 2D and 3D models of NETs to evaluate the impact on cell growth and survival. We will also explore the impact of 161Tb on spheroid transcriptome, this will allow the identification of distinct and overlapping effects of varied therapeutic doses of 161Tb.

Actithera is a seed stage Radiotherapeutics company that has developed several strategies for selectively prolonging the tumor retention of radioconjugates. FAP (Fibroblast Activation Protein) is currently one of the most important theranostic targets because it has nearly pan-cancer potential due to its high expression in more than 90% of solid tumors, but low or no expression in normal resting cells. FAP has gained notoriety about how difficult it is to achieve compounds that show sufficiently long tumor retention in FAP-expressing tumors to be considered as successful therapeutics. Several compounds that have advanced into clinical trials have shown significant promise as diagnostics due to their specificity for FAP-expressing tumors and low background, but not as therapeutics because they clear out of tumors within hours instead of days. Actithera has applied its proprietary approaches in the design of novel FAP tracers with the goal to find compounds with the appropriate pharmacokinetic profile for therapy. Two prototype Actithera compounds, have shown clearly i) superior tumor retention; ii) tumor/normal ratios and iii) efficacy over three key clinical stage comparators in vivo. These compounds show a lot of promise and differentiation over the state of the art in the FAP field. One of them, ACT-3-19 is selected for clinical development. The objective of this application is to test ACT-3-19 and a second compound in a clinically relevant orthotopic mouse pancreatic cancer model and compare the effects of three important medical radionuclides: two beta emitters: Lu-177 and Tb-161 and one alpha emitter Ac-225. No such studies have been published before, especially in the presence of intact immune system, as we are planning to do here. Such a study will guide the clinical development of ACT-3-19 but also of other FAP-targeting agents and will provide significant new insights to the theranostic community.

Targeted alpha therapy (TAT) is a cutting-edge form of radiotherapy that uses alpha particles, which are highly effective in destroying cancer cells. A key element in this approach is radium-223, a radioactive isotope that emits alpha particles and has shown promise in treating certain cancers, such as prostate cancer. However, a major challenge is ensuring that radium-223 remains securely bound to the delivery compound, preventing it from harming healthy tissues. The goal of this research is to design new macrocycles that can tightly hold radium-223, ensuring it is delivered safely to cancer cells without harming healthy tissues. The current gold-standard chelator, macropa, faces stability issues when linked to vectors—molecules that guide the treatment to the cancer cells— leading to the release of radium in the body. Our proposed macrocycles are designed to address this limitation by incorporating structural elements that enhance rigidity and stability. By conducting detailed studies on the properties of these new compounds, we aim to identify the most stable candidates for further development. Ultimately, this research could lead to more effective and safer cancer therapies, expanding the options available for patients undergoing targeted alpha therapy. This project brings together expertise in chemistry and radiopharmaceuticals from leading research groups at the University of Brest and the Comprehensive Cancer Center Eugène Marquis in Rennes. Through the partnership with the PRISMAP network, we are laying the groundwork for future clinical applications, with the potential to transform cancer treatment.

Targeted radionuclide therapy is nowadays well accepted for the management of metastatic cancer patients. Its use is envisioned at the upfront of treatment algorithms as exemplified with the recent data presented at ESMO24 in prostate cancer and recent pilot studies, for better efficacy and improved survival. However, to achieve cure, the adjuvant use of TRT should be investigated to eradicate disseminated tumor cells and cancer stem cells (CSC) that are thought to be at the origin of cancer recurrence and metastases. Auger-emitters are well suited to achieve this objective considering their low range minimizing side-effects. Moreover, their high local deposit of energy make them relevant radionuclides to eliminate single cells or microcluster of cancer stem cells. Palladium-103 is an Auger-emitter which has gained recent interest for TRT. 103Pd decays (T1/2 = 16.991 d) by electron capture into rhodium-103m (103mRh), which in turns decays (T1/2 = 56.11 min) through isomeric transition into stable 103Rh. The contribution of 103mRh is about 45% of Auger-electron emitted by the decay chain 103Pd/103mRh and must be also be retained in tumors for effective TRT and reduced toxicity. Thus, 103Pd/103mRh stands as an in vivo generator that must be correctly captured to develop radiopharmaceuticals designed for single cell/micrometastases killing. Therefore, the main objective of the project is to develop chelates able to coordinate both 103Pd and 103mRh

Gathering three academic research teams from Dijon, Strasbourg, and Paris-Saclay in addition to the French biotechnology company Syndivia, the TheraTerb project seeks the development of innovative radiopharmaceuticals (RP) for the treatment of metastasized castration-resistant prostate cancer (mCRPC). The proposed drugs involve the multipurpose Tb-161 radionuclide (RN), a beta– emitter that provides also low-energy conversion (CE) and Auger electrons (AE) and gamma rays, the latter for SPECT investigations. The advantage of Tb-161 over Lu-177, the current RN used to cure mCRPC in Lu-177-PSMA-617 (Pluvicto), is the CE/AE emission, which allows it to kill single or small cell aggregates before they develop to such an extent that PET or SPECT imaging can detect them. The originality of our RP design rests on the replacement of the ubiquitous DOTA-type bifunctional chelators by open-chain ligands able to sequester rapidly Tb-161 at room temperature, unless DOTA that forms stable and inert metal complexes only under denaturing conditions. TheraTerb addresses mCRPC as oncological target, not only because of its implication in male death rate, but also in order to test a new PSMA-specific mAb (D10) developed by Syndivia. D10 shows enhanced tumor penetration and morover targets a novel epitope, distinct from the one of Pluvicto, circumventing the nephrotoxicity and salivary gland toxicity of the latter. Syndivia will apply its GeminiMab™ technology, an original enzyme-free bioconjugation method involving a FDA approved conjugation site, with precise control of drug-to-antibody ratio, which will guarantee minimal perturbations of the pharmacokinetic properties of the antibody-drug conjugate with respect to the native mAb. In vitro and in vivo evaluation of the radiopharmaceuticals will include immunoreactivity assays with C4-2 cell lines before collecting µ-SPECT-CT images, biodistribution and dosimetry data for mice bearing xenografted mCRPC tumors.

When using radionuclides in nuclear medicine, it is essential that their activities are measured accurately and that all measurements are realized on a common international metrological basis. Nuclear decay data are particularly important when using radionuclides for medical applications. Half-lives as well as photon emission intensities are essential for activity determination, decay corrections, dose estimates, radiation protection purposes, the treatment and disposal of related radioactive waste and other potential applications such as quantitative imaging. In addition, a determination of remaining radionuclide impurities after purification is important as well. The overall aim of the project is to establish measurement techniques for the determination of the activity of 175Yb and to study its nuclear decay properties in detail. The specific objectives are: (1) Development of primary activity standardization techniques to determine the activity concentration (activity divided by mass of solution) of 175Yb with high accuracy and low uncertainty (2) Determination of the 175Yb half-life by means of long-term measurements (3) Determination of decay scheme parameters The above-mentioned objectives will be complemented with calibrations of instruments (mainly ionization chambers) for secondary activity standardization at PTB. In this way, the results of primary activity standardization can be conserved, and the established calibration factors will make future calibrations by means of ionization chambers possible.

In recent years therapy with Auger electron emitters has been proposed as a promising strategy for selective cancer treatment. Unfortunately, commonly used Auger emitters like 111In, 125I, 67Ga and 201Tl are also effective γ-emitters and emitted γ quanta strongly contribute to healthy organ toxicity. 103mRh is one of the most promising radionuclides for Auger radiotherapy. The decay of 103mRh is associated with the high Auger electron yield compared to its emissions of photons (X-rays). Unfortunately short half-life of 103mRh (t½=56.11 min) makes it difficult to therapeutic use but can be "extended" by using 103Pd, the parent radionuclide for 103mRh. Considering all this, we propose new idea to deliver the 103mRh to the cell nucleus by using an in-vivo 103Pd/103mRh generator conjugate. In our approach 103Pd radionuclide will be complexed with derivatives of tetraaza ligands and connected to two commonly used vectors in nuclear medicine: trastuzumab monoclonal antibody and octreotide peptide. Trastuzumab binds specifically to HER2 receptors on breast and ovarian cancers, and octreotate exhibits an affinity for somatostatin receptor on neuroendocrine tumors. Both vectors have the ability to be highly internalized into the cell cytoplasm. The obtained 103Pd labelled radioconjugates should accumulate in cancer cells and active pass through the cell membranes. Afterward, as a result of nuclear decay 103Pd→103mRh, the Auger electron emitter 103mRh will be partially released from the 103Pd complex and in the chemical form of 103Rh_aq^(3+) can transfer through the nuclear membrane and bind to the DNA, inducing cytotoxic effects by Auger electron emission. If we achieve positive results in this project, we can proceed to the next phase, which includes extended preclinical and clinical trials. Auger electron therapy has the potential to significantly enhance the effectiveness of treating aggressive breast, ovarian, and neuroendocrine cancers while minimizing side

Despite medical advances in prevention, early detection and treatment, cancer remains a leading cause of death worldwide. Within this context, targeted radionuclide therapy is a potent approach, especially for cancer patients that have exhausted all other therapeutic options. Among the radionuclides available in the PRISMAP portfolio, silver-111 (111Ag, t1/2 = 7.47 d) has optimal theranostic features possessing a medium-energy β− (Eβ−, max = 1.04 MeV) and a low-energy γ (E? = 245.4 keV, I? = 1.24 %; E? = 342.1 keV, I? = 6.7 %) emissions that could be exploited for theranostic application in badly perfused metastases where cancer cells need to be targeted indirectly. From a chemical point of view, silver belongs to the transition metals of the 11th group and shares some chemical features with copper, the lightest element of the group. Unfortunately, in contrast to copper, its coordination (radio)chemistry, in particular in biological systems, is characterized to a lesser extent. The overarching aim of this application is to develop suitable chelators able to stably coordinate silver and exhibiting high inertness in physiological condition in order to trigger the possible use of 111Ag for clinical applications. The aim will be pursued through an interdisciplinary approach ranging from theoretical calculation to fundamental coordination chemistry. Radiolabeling with 111Ag and stability assays under simulated biological conditions will be conducted as well. In particular, for the estimation of the stability in human serum, we plan to use Perturbed Angular Correlation (PAC) spectroscopy as potential alternative and additional tool to the standard techniques usually applied. Finally, the biodistribution of the 111Ag-complexes with the most promising chelator(s) will be evaluated in murine models and compared to the physiological biodistribution of free111Ag.

In recent years, molecular radiotherapy has revolutionized the field of cancer therapy for neuroendocrine- and prostate cancer. By attaching a therapeutic radionuclide to a cancer-targeting molecule, the radiation source is transported to, and accumulated on, the cancer cells. This creates a local radiotherapy treatment whilst sparing normal tissue. In the present project, we aim to bring our newly developed cancer-targeting radiopharmaceutical to patients with aggressive cancer forms such as Anaplastic Thyroid Carcinoma (TC), advanced Head & Neck Squamous Cell Carcinoma (HNSCC) and Non-Small Cell Lung Cancer (NSCLC). In a project supported by The Swedish Cancer Society (Cancerfonden), and Sweden's Innovation Agency (VINNOVA), we have designed a novel 177Lu radiolabeled CD44v6-targeting antibody (AKIR001). The project was recently awarded 10 MSEK (approx. 1 M€ ) by The Swedish Cancer Society, focusing on first-in-man studies in patients with target-positive solid cancers with the 177Lu-labeled antibody. In this program we aim to expand the therapeutic options through preclinical assessments of 161Tb as an alternative and potentially superior radionuclide to 177Lu for AKIR001. We believe the properties of 161Tb, with the combined beta and auger emission, might prove superior to 177Lu. As the antibody has already been optimized for DOTA chelation, we expect the radiolabeling with 161Tb to be feasible. In the project, we will start by optimizing the 161Tb radiolabeling of AKIR001, followed by binding characterizations in vitro on cancer cell lines. We will then move on to therapeutic assessments in e.g. 3D tumor spheroids. If time allows, we will also assess combinations with potential radiosensitizing agents (e.g. HSP90 inhibitors), an/or radiolabeling of additional cancer targeting molecules. If successful, in vivo studies in tumor bearing mice will then be performed, assessing biodistribution followed by dosimetric assessments and therapy studies.

It has already been proven, that 225Ac-mcp-D-PSMA exhibit higher tumor uptake compared to 225Ac-PSMA¬617 and also to 225Ac-mcp-M-PSMA combined with a fast renal excretion and neglectable off-target accumulation, due to higher molar activities and doubling of the PSMA-binding motif connected to one macropa chelator. Further, shorter reaction times at ambient temperature lead to no degradation of the precursor peptide during radiolabeling and even permits the radiolabeling of antibodies. Ultimately, macropa is also capable of forming stable complexes with 203/212Pb at ambient temperature and very low amount of precursor (down to 0.1 µmol/L). This would produce high molar activity radiopharmaceuticals for theranostic purposes. Precursor and optimized radiolabeling protocols for 203/212Pb are already established. Within this project, the automation will be implemented on a commercial GAIA synthesis module from ElysiaRaytest, which is suitable for the radiolabeling of peptides and small molecules in aqueous medium with radiometal nuclides, and in particular with 68Ga3+. This module is equipped with the LUNA extension module being developed to allow also labeling with therapeutic β–-emitters such as 177Lu3+. No synthesis modules for the production of 212Pb-based radiopharmaceuticals are not yet commercially available. The clinical production for 203/212Pb-labeled radiopharmaceuticals is established manually so far. The translation to a GMP-conform production of the new radiopharmaceutical 203/212Pb-mcp-D-PSMA should be performed within the time frame of one year. Patient application could also be performed in the coverage of an individual theranostic treatment.

Neuroblastoma (NB) is the most common extracranial solid tumour of childhood, and has an international incidence rate averaging 10.1 per million children aged 0-14 years – with approximately 90% of cases occurring in patients under the age of 5. Of all NB cases, ~50% of patients are categorised as high-risk, with a three year event-free survival currently just over 50%. High-risk neuroblastoma is considered radiosensitive, and therefore molecular radiotherapy has an established place in the management of refractory and relapsed disease. For example, 131I-mIBG beta (β-) particle emitting radiotherapy in NB has a response rate of 14%. Alternatively, 177Lu-DOTATATE radiotherapy, has also been evaluated in clinical practice, and is currently the focus of clinical trials. However, target expression for these agents has been shown to be heterogeneous in NB patients; resulting in incomplete targeting of the radiation to tumour cells and deposits. An alternative therapeutic target in NB is di-sialo-ganglioside (GD2) which is a small sialic acid containing glycolipid that is highly expressed in NB, compared with normal tissues. In fact, an FDA & EMA-approved monoclonal antibody targeting GD2, Dinutuximab Beta (DB), has vastly improved survival in high-risk neuroblastoma patients. We have recently begun to validate 177Lu-labelled DB (177Lu-DB) as a beta-emitting molecular radiotherapeutic against GD2-positive NB. However, beta therapy can be less effective at treating smaller tumours and can also cause damage to tumour-adjacent healthy tissue. Alternatively, alpha-emitting (α) radionuclides can be more effective at treating isolated cancer cells and/or micrometastases, causing greater DNA damage than beta-therapy. In particular, the alpha emitting isotope 225Ac has previously shown clinical efficacy, and is seen as a prime candidate for radioimmunotherapy. Hence, in this project, we wish to develop a GD2-targeted alpha radioimmunotherapy using DB conjugated with 225Ac.

Auger electron (AE)-emitting radionuclides have gained increasing interest for their potential applications in treating metastatic disease. AE therapy is deemed to be equally efficient as α-particles, with the benefit of limited normal-tissue toxicity. Still, basic research into the radiobiological and dosimetric mechanisms leading to AE therapeutic effects is a work in progress. The availability of 'pure' AE emitters, such as Lanthanum-135 (135La), enable us to study the absorbed dose – response relationship which can guide future radiopharmaceutical development. Due to 135La's medium energy and yield AE's, if falls within the range of AE's emitted by other promising radionuclides such as Terbium-161. In this proof-of-principle study, we will assess the differential effects of internalizing (agonist) vs. non-internalizing vectors (antagonists) labeled with 135La in in vitro models of neuroendocrine tumors (NETs). Preparatory work includes the calibration of all quantitative equipment prior to in vitro binding and internalization studies which serve for dosimetric calculations of the absorbed dose in different cell compartments. Radiobiological effects and dose-response relationship of 135La-agonist and antagonist treatment will be further evaluated by standardized methods.

Background Gastrin Releasing Peptide receptor (GRPr) represents an innovative imaging target since it is overexpressed on a variety of human tumors. GRPr-based radioligands, mainly antagonists, have shown great promise for diagnostic imaging of GRPr-positive cancers, such as prostate and breast. It has been proven preclinically but also in the clinical settings that the late imaging of GRPr-based radioantagonists is beneficial since they better localize primary and recurrent disease due to the faster washout of the radioactivity from the background organs compared to the tumor. If GRPr-ligands are labeled with the promising theranostic pair 44/47Sc, the created radiotracers would be of utmost importance since patients could benefit not only from the advantages of the late imaging but also from the theranostic approach. Aim The overall aim of the project is to create "theranostic twins" from newly developed GRPr peptide antagonists, suitable for labelling with 44Sc (diagnostic radionuclide) and 47Sc (β- therapeutic radionuclide) and to be used for the in vivo imaging and therapy of GRPr positive tumors. Expected results In terms of affinity, improved GRPr-specific ligands will be developed, suitable for labelling with 44/47Sc. The extensive in vitro and in vivo evaluation using a variety of cell lines and organoids will provide valuable data with respect to their binding properties and their in vivo performance both as diagnostic and therapeutic agents. Impact It would be a great progress if nuclear medicine could have a diagnostic imaging method that would not miss any prostate-tumor, detect a variety of other GRPr-positive tumors as well as more metastases than current methods allow. In addition, the generation of the theranostic pair would be the basis for a more successful targeted radiotherapy.

Tumor heterogeneity and in particular intrapatient and intratumoral heterogeneity can lead to false (negative) diagnoses and thus, their identification is crucial to prevent inadequate patient treatment. A combined use of radiolabeled tracers specifically binding to different target structures of the tumor is supposed to improve lesion detection and characterization (e.g. aggressive versus non-aggressive). Provided that the signals emitted by the different probes remain distinguishable, this dual-tracer-based method might allow for non-invasive estimation of intrapatient and intratumoral heterogeneity. For a first proof-of-concept study we aim at the combination of 18F and 44Sc, since recent studies on a VECTor6 system (MILabs) demonstrated the distinguishability of two different PET nuclides by using a system of pure and non-pure β+ emitters. For setting up the imaging procedure on our VECTor4 scanner system, phantom measurements with 18F (pure β+ emitter) and 44Sc (non-pure β+ emitter) are needed to investigate the performance of the detectors. This includes measurements with different collimators, acquisition modes (spiral or multi-planar) and acquisition of activities of different magnitudes (e.g. 1 MBq, 10 MBq and 50 MBq of 44Sc or 18F or mixed) for each collimator and acquisition mode. In case of a successful technical implementation, further studies (beyond this PRISMAP project) are planned to investigate the transferability to in/ex vivo studies, as we aim at the development of suitable dual-tracer PET applications with high relevance for clinical purposes and personalized medicine.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected using current clinical tools, that progress into lethal lesions during the course of the disease. We propose to develop an exosome-based theranostic nanoplatform for the detection and treatment of lung micrometastasis, using tumor-derived exosomes (EXs) as natural delivery vehicles of positron (64Cu) or beta-emitting (67Cu) radionuclides for imaging diagnosis by PET and targeted radionuclide therapy. This proposal takes advantage of the current breakthrough knowledge regarding the crucial role of EXs on specific metastatic organotropism and from the advances of molecular imaging technology and radionuclide-based therapies, that have been employed with high effectiveness in oncology, paving the way towards individualized medicine. We have already optimized the reaction conditions for the radiolabeling of EXs with Cu-64 with high efficiency and purity. The in vivo studies showed the specific accumulation of the radiolabeled exosomes in lung metastatic lesions with 2-3 mm in diameter as well as in the primary tumor, confirming its potential as PET imaging tracers. This work was recently published (https://pubmed.ncbi.nlm.nih.gov/36316233/) Following up on this work, we intend to exploit the use of EXs as a theranostic tool by replacing 64Cu by the beta-emitting 67Cu for targeted radionuclide therapy. The therapeutic efficacy will be evaluated in the lung metastatic mouse model. With this work, we expect to develop a [64Cu/67Cu]EXs theranostic tool highly effective in the early detection and treatment of lung metastasis, which represents a major challenge in OS patients' management with increased clinical translational potential. We routinely produce 64Cu using our cyclotrons at ICNAS. We are applying for the supply of 67Cu from the PRISMAP network.

Patients with glioblastoma (GBM) have a poor prognosis following standard therapy (median survival is 12-15 months), with a 5-year survival rate of only 3-5%. Currently, the standard GBM treatment includes maximal resection (complete resection is achieved extremely rarely due to the diffusely infiltrative nature of these tumours) followed by radiotherapy with concomitant and adjuvant systemic therapies e.g., temozolomide. Despite these aggressive regimens, most patients become refractory to treatment and succumb to disease. Therefore, there is high unmet medical need for new treatment paradigms that lead to more durable remissions. EGFR is mutationally activated in approximately 50% of GBMs which correlates with a more aggressive disease course. We hypothesise that locoregional administration of molecularly targeted radionuclide therapy with highly cytotoxic radioisotopes (e.g. 161-terbium) associated to EGFR specific vector will selectively induce cell death in EGFR-positive GBM cells, while limiting toxicity in the surrounding normal tissues. Furthermore, we postulate that this therapeutic approach will decrease the immunosuppressive GBM tumour microenvironment (TME) and enhance the sensitivity to checkpoint inhibitors.

Radioligand Therapy (RLT) has fast become an established approach in the fight against cancer. Previously, several PARP inhibitors coupled with different radionuclides have been developed by us and others, for imaging and RLT. However, the comparative efficacy and radiobiological effect of these compounds has not been systematically explored. We do not fully grasp the biological effects that are triggered, or which emitters are more useful in RLT with intracellular targets. Should this be accomplished, together with a more refined understanding of radiation dose, dose rate, and radiation dosimetry, then patient selection, and the efficacy of RLT will be much improved. Therefore, we propose a systematic comparison of PARP inhibitors labelled with (211At, 225Ac), beta (161Tb, 177Lu), or Auger-electron emitting radionuclides (123I, 125I, 161Tb). Our eventual goal is to identify which offers the greatest radiotoxicity with the fewest undesired side effects. We will synthesise a series of radiolabelled versions of the PARP inhibitor, olaparib. Subsequently, we will expose a panel of human cancer cell lines, expressing different PARP levels, to these radiolabelled PARP inhibitors. The objective is to determine how these compounds differently affect cell survival and identify which signalling pathways are triggered by them. In a follow-up project, we will also test their efficacy in vivo and determine normal tissue toxicity. With this project we aim to unveil new strategies to develop RLT and explore the differences between different radionuclides, using the same vector. This will undoubtedly open new pathways for optimised RLT selection which will have a significant impact by enhancing the selection of RLT drugs, establishing the foundational knowledge for RLT development, and advancing the development of new RLT drugs that could eventually be integrated into patient care.

The aim of this project is to characterize the quantitative performances and image quality achievable in 155Tb SPECT images in the presence of a certain level of contamination by 156Tb in a preclinical SPECT scanner (ALBIRA II) configuration. When 155Tb is produced in a commercial or research cyclotron via proton or deuteron induced reactions, it is invariably accompanied by contaminants, notably 156Tb. The high energy gammas emission from 156Tb will contribute to pollute the 155Tb signal, with consequent detriment in the resulting SPECT image quality and quantification. This study will compare the achievable SPECT image quality and quantitative performances based on NEMA NU4 phantom experiments employing both pure 155Tb solution and 155Tb/156Tb mixed solution (155Tb/156Tb activity concentration ratio = 10:1). Using the GATE software, preliminary computed Monte Carlo simulation of the NEMA phantom acquisition in the ALBIRA II pre-clinical system guided the selection of the most appropriate collimator geometry (i.e. the thickness) and acquisition energy window. To reduce the collimator penetration by the high energy gammas emitted by the 156Tb, at an acceptable level, a 5 mm tungsten collimator is needed. The anticipated acquisition energy window is 70-110 keV. Similar experimental setup has already been successfully used by Müller et al. (with multi pinhole collimator though) but without quantitative assessment of the 156Tb contribution. The effects of the 156Tb contaminant on quantitative accuracy will be investigated, as well as its impact on image quality metrics such as coefficient of variation, contrast to noise ratio, hot and cold contrast, and recovery coefficients in cylindrical phantom inserts. This study will provide an upper limit for the 156Tb contamination compatible with quantitative 155Tb SPECT imaging relevant for preclinical and possible future clinical applications.

Terbium offers 4 clinically-relevant radioisotopes with a wide range of T1/2, which could be used in diagnostic (PET/SPECT) and therapy (alpha, beta–, and Auger), making this radionuclide a multipurpose theranostic element. The project TerbCheNuM focuses on 155Tb (5.32 d T1/2) that can be used in SPECT imaging and Auger therapy. The current radiopharmaceuticals incorporating 155Tb are made of DOTA-peptide bioconjugates. They tolerate the high temperatures used for insertion of the metal into the DOTA cavity, which is not the case of mAbs bioconjugates. The aim of TerbCheNuM is therefore to develop so far unavailable terbium-specific bifunctional chelators that can be radiolabeled in mild conditions (25 °C) while keeping the high stability and inertness of the DOTA complex. Unlike typically hard cations, such as Fe3+, which display a strong affinity for chelators based on negatively-charged oxygen donor atoms, lanthanide trications such as Tb3+ require chelators combining these donor atoms with relatively soft neutral nitrogen donor atoms, as in DOTA-based chelators (4 O–, 4 N). The slow complexation kinetics of the latter being due to its macrocyclic topology, the proposal rests on the design, synthesis and evaluation of mixed (O–, N) chelators displaying either linear or branched topologies, which give rise to faster complexation reactions. The former will be obtained by extension of the well-known DFO chelator with a fourth all-nitrogen ligand subunit; the latter will be based on pyridylcarboxylate-substituted diamines. The chelators able to complex Tb3+ in mild conditions will be radiolabelled, which will allow us to compare their stabilities in different media, and to select those that will be bioconjugated to trastuzumab, an antibody specifically targeting HER2. The radiopharmaceuticals will be used for investigations on SK-BR-3 cell lines firstly for assessing their stability in vitro, secondly for tumour imaging and biodistribution investigations in vivo.

The effectiveness of the treatment in internal radiotherapy using radioisotopes with high linear energy transfer emissions is primarily determined by the emission type; however, indirect effects inflicted in cancer cells and tumor microenvironment contributes substantially to trigger apoptosis, necroptosis or other cell death forms thus improving the therapeutic outcome. Synergistic effect of molecular radionuclide therapy and anticancer therapy using DNA-dependent protein kinase (DNA-PK) inhibitors is sought, when the DNA damage response modulator impairs a pathway involved in the repair of the damage induced by radionuclide therapy. This proposal aims to explore the DNA damage-inducing anticancer therapies using 4 of the most promising therapeutic radionuclides (64Cu, 67Cu, 211At, and 161Tb), also picturing the apoptosis, repairing mechanism, relevant genes expression, and stress response. We propose to preliminary investigate in vitro, on relevant tumor cell lines, the biochemical modifications in cell viability, cytotoxicity, proliferation, cell cycle, and apoptosis inflicted by MRT monotherapy as well as the combinatorial approaches of MRT and DNA-PK inhibitors. DNA damage and stress responses extensive insights will allow for a rational combination design, optimal dosing and sequencing.

Expression of the integrin αVβ6 is upregulated in a range of difference carcinomas, including pancreatic ductal adrenocarcinoma and breast cancers, whilst having low expression in healthy adult tissue. Increased expression often correlated with poorer prognosis, which makes αvβ6 an attractive target for molecular imaging and peptide receptor radionuclide therapy (PRRT). The peptide A20FMDV2 has high selectivity and affinity for αvβ6, and has been radiolabelled with a range of isotopes and across a range of structural modifications. Currently, there are no clinically approved αvβ6-targeted radiotherapeutics, despite its importance as a target. There are ongoing clinical trials for imaging αvβ6, but none for targeted alpha therapy (TAT). The use of α-emitting radionuclides in therapy is attractive for treating micrometastases and isolated cancer cells due to the short range of the emission and the high linear energy transfer (LET) (a factor of up to 10^3 higher than the LET of β-emitters), which provides a higher chance of causing irreparable double-stranded DNA breaks per emission. Lead-212 has been used preclinically and in clinical trials as an in vivo generator of bismuth-212, an α-emitting radionuclide, and has shown anti-tumour efficacy. The shorter half-life of lead-212 compared to the clinically used α-emitter actinium-225 allows for administration of higher activity doses, and is suitable for TAT using vectors with a short biological half-life, such as peptides. Also, lead-203 can be used as an imaging surrogate for lead-212, making lead-212 the only α-emitter with a direct theranostic isotopic partner. We aim to use this project to utilise lead-203 for the assessment of a range of A20FMDV2 compounds as potential targets for development of their lead-212 analogues for future studies.

Targeted alpha-therapy (TAT) is an emerging and powerful approach for treating cancer. It uses alpha-emitting radionuclides to specifically target and destroy cancer cells, particularly in cases where therapeutic options are limited. Astatine-211 (211At) TAT has shown promising results in preclinical studies and is currently being evaluated in clinical trials for the treatment of various cancers. In the case of glioblastoma (GBM), translating preclinical research into clinically relevant human therapy is challenging due to differences in cell types, receptors, resistance, and the tumour microenvironment between preclinical and clinical models. To the best of our knowledge, some clinical studies in humans were completed. One of them demonstrated that TAT with astatine-211 conjugated to the chimeric monoclonal antibody ch81C6 administered into a surgically created resection cavity was feasible and safe, showing encouraging overall survival results. Glioblastoma cells overexpress the neurokinin-1 receptor (NK-1), which binds the natural ligand Substance-P (SP). A targeting system based on [211At]At-SP for TAT in GBM has never been addressed in a clinical study. We plan to perform a pilot study to determine the feasibility of a randomized controlled trial (RCT) on the efficacy and safety of [211At]At-SP as adjuvant therapy for recurrent human glioblastoma. We intend to go beyond the state of the art by using data from this pilot study to improve the design of a larger-scale, statistically powered RCT. Thus far, studies have shown that [211At]At-SP targeting NK-1 receptors are the most promising candidates for a breakthrough in GBM treatment. There are currently no ongoing trials identified proposing the use of this radiopharmaceutical for theranostic purposes in patients with GBM.

Targeted alpha-therapy (TAT) is an emerging and powerful approach for cancer treatment that uses alpha-emitting radionuclides to target and destroy late-stage cancers cells for which therapeutic options are limited. Ac-225 TAT has shown promising results in preclinical studies and is currently being evaluated in clinical trials for the treatment of various cancers, including leukemia, prostate cancer, and neuroendocrine tumours. However, the poor understanding of Ac-225 coordination chemistry creates challenges for the development of suitable chelation strategies for this ion. Thus, the ideal chelator should exhibit fast metal-complexation kinetics, selectivity for the radionuclide, high thermodynamic stability and high in vivo stability. The main goal of this proposal is to test novel chelators based on 6-aminoperhydrodiazepine and aminomethylpiperidine scaffolds bearing three picolinate pendant arms to obtain nonadentate and octadentate ligands for Ac-225 labelling. Ac-225 labelling test will be performed at MRI-TUM in order to find the best conditions to obtain high labelling yields. The stability in physiological conditions and in human serum will be also investigated both on the cold La(III) and the Ac-225 complexes to evaluate their potential in vivo stability. Furthermore, a bifunctional chelator will be conjugated to an antibody-derived molecule and/or to peptides for Ac-225 labelling and in vivo testing. The advantages of this project are: i) use of a semi-rigid scaffold coupled to picolinate pendants for room temperature Ac-225 labelling, ii) construct a bifunctional chelator for bioconjugation, and iii) developing a novel stable probe for TAT; the proposal is therefore highly innovative, and will allow the assessment of the potential of these tracers for clinical translation.

Targeted radionuclide therapy has proven to be a valuable therapeutic option for metastatic patients. Novel therapeutic radionuclides emitting short range particles are on the horizon and are expected to provide better efficicacy than 177Lu. However, the current xenografted mice model has severe limitations to truly evaluate the effects of these short range therapeutic emitters due to the absence of isolated metastases. Zebrafish is an emerging model in oncology that allow the implantation of human tumor cells (cell lines or patients' cells) that can next metastazed to zebrafish organs. Therefore, it is possible to obtain in the same model a primary tumor and isolated metastases. In this project we aimed at using zebrafish embryo as a novel model to better evaluate the efficacy of short range emitters at a multicellular level and on isolated cells. 161Tb and 155Tb will be used as demonstrative examples.

Immunotheranostics is today recognized as a game-changer in the patient-care in oncology for diagnosis, follow-up and treatment. These new approaches combine the unparalleled targeting potency of antibodies with dedicated radioisotopes, either for imaging diagnosis (i.e. with positron emission tomography - ImmunoPET) or for vectorised internal radiotherapy (radioimmunotherapy). ScandAL will boost such approaches in synergizing three pivotal parameters that are (i) the theranostic pair of isotopes, (ii) the radiolabelling strategy, and (iii) the biological vector. To match the slow pharmacokinetics of full antibodies (over several days), zirconium-89 (half-life 78 hrs) is usually used for ImmunoPET imaging but leads to high radiation exposure (gamma ray at 909 keV). It requires moreover for patients two visits at the hospital because imaging sessions are scheduled three to five days after tracer injection to obtain the optimal tumour accumulation of the radiolabelled antibody. To unlock these limitations, ScandAL proposes to use a minibody (i.e. antibody fragment) targeting one immune-check point, PD-L1, conjugated with the emerging scandium-43 positron emitter that will be introduced via an original disulfide rebridging approach. ScandAL aims at highlighting the superiority of this approach over standard methods in force today. Antibody fragments (Fab, diabodies, minibodies…) have faster pharmacokinetics that fit the 4 hours half-life of scandium-43 thus leading to a decrease of radiation burden and duration of examinations. Moreover, disulfide rebridging is a site-specific approach to radiolabel antibody fragments affording a defined radiolabelled compound contrarily to standard random labelling methods that lead to uncontrolled mixtures of species. ScandAL will deliver a proof of concept in diagnostic imaging that could be extended to therapy with the use of scandium-47, a beta-emitting counterpart.

Notwithstanding the decline in mortality rate observed in the past decade, cancer remains one of the main causes of death worldwide. As such, the need for more efficient diagnostic methods and novel therapy alternatives has propelled important advances in cancer research. Targeted radionuclide therapy (TRT) is an anticancer therapeutic modality particularly attractive for disseminated disease with potentially fewer side effects than conventional radiotherapy. The aim of this project is to evaluate the radiobiological effects of 64/67Cu-based agents for TRT of resistant cancers. We intent to go beyond the state-of-art by performing a thorough radiobiological evaluation in advanced cell models of prostate and ovarian cancers. This project gathers a team of Portuguese and French researchers with complementary know-how in radiopharmaceutical development and preclinical evaluation and offers possibilities for advanced training of young researchers from both countries.

Fibroblast activation protein (FAP) is a cell surface marker of cancer- associated fibroblasts (CAFs) in most sarcomas and in > 90% of carcinomas. Together with its negligible expression in most other tissues, this makes FAP a nearly-universal biomarker of tumors. During the past years, diagnostic and therapeutic targeting of FAP with so-called 'FAPIs' has attracted strong attention from nuclear medicine/oncology specialists. Noteworthy, all FAPIs owe their remarkable tumor homing potential to a potent and selective FAP- binding subunit: UAMC1110, reported by the applicants of this proposal. Because FAPIs require further optimization of tumor residence time, we aim to link multiple FAPI subunits to gold nanoparticles (AuNPs). In this way, we hope to obtain FAP-targeting AuNPs with unprecedented FAP affinity and tumor residence, due to the 'multivalency effect'. The nanoparticles will be investigated as cancer theranostics in a mouse model of colorectal cancer.

The prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer tissues at considerably higher levels compared to healthy organs. Therefore, PSMA has emerged as an attractive target for molecular imaging and especially targeted radionuclide therapy (endoradiotherapy) of metastatic castration-resistant prostate cancer (mCRPC), given the example of [177Lu]Lu-PSMA-617. We recently described the synthesis and in-depth characterization of a range of novel PSMA radioconjugates with different pharmacokinetic properties and biodistribution profiles for targeted alpha therapy with 225Ac. Thereby, we identified two promising candidates with a remarkable tumor accumulation, favorable tumor-to-background ratios and adequate blood circulation times, which are worth to be evaluated in a preclinical targeted alpha-radiation therapy study using a murine prostate cancer mouse model as the next step. We are therefore applying for the supply of the required activity of 225Ac by the PRISMAP network. Specifically, we plan to perform an activity-dependent radionuclide therapy study using a murine subcutaneous xenograft model for the assessment of the therapeutic potential and the treatment efficacy of both 225Ac-labeled conjugates. The proposed project represents a key requirement for the clinical translation of these novel 225Ac-radioconjugates and is therefore essential for the continuation of our research in the field of PSMA-targeted alpha therapy aiming at first applications in prostate cancer patients.

Radioisotopes of the lanthanides (Ln) and actinides (An) are being extensively investigated for their application in diagnosis and systemic radiotherapy in nuclear medicine. Radioisotopes of terbium have applications in imaging and radiotherapy. In particular, Tb-161 has shown superior radiotoxicity compared to Lu-177 in recent studies. Alpha-emitting actinide radioisotopes, Ac-225 and Th-227, have demonstrated exceptional therapeutic efficacy when tethered to targeting vectors. To date, most radiopharmaceuticals based on Ln and An radioisotopes use DOTA as the chelating agent for the radiometal. Whilst DOTA forms highly stable complexes with these radiometals, radiolabelling/complex formation often requires high temperatures, which are not compatible with many biological agents – for example, antibodies for use in systemic radioimmunotherapies. We have designed and synthesised a library of hybrid chelators based on macrocyclic cyclen or cyclam groups, each bearing four coordinating hydroxypyridinone motifs, enabling complexation of large Ln and An ions, for application in nuclear medicine. These chelators have the potential to allow highly selective, sensitive biological molecules to be radiolabelled under mild conditions, for development of novel Ln and An radiopharmaceuticals. We will test radiolabelling of these new chelators with Tb-161, Ac-225 and Th-227, and assess the stability of new radiometalled chelators in serum. We will synthesise bifunctional derivatives and PSMA-targeted bioconjugates of the best performing chelators, and radiolabel these with Tb-161, Ac-225 and/or Th-227. Finally, we will show proof-of-principle of the utility of these new chelator platforms, by testing the new radiolabelled derivatives in vitro and in vivo, in prostate cancer models.

Recently, prostate-specific membrane antigen (PSMA) emerged as specific target for both molecular imaging and radionuclide therapy in prostate cancer (PC). Indeed, radionuclide 177Lutetium PSMA-targeted treatment has been recently approved by FDA in US and is expected soon in EU, for metastatic castration-resistant prostate cancer (mCRPC). A potentially more effective and less toxic therapeutic alternative to 177Lu-PSMA is 161Tb-PSMA. Our long-term goal is to provide the evidence for 161Tb-PSMA to be transferred into the clinicals to target the micrometastatic disease as early as at the biochemical recurrence. We plan to obtain the objective of this application by pursuing the following specific aims: 1. Evaluate the efficacy of 161Tb-PSMA and 177Lu-PSMA in cell lines, subcutaneous and spontaneous orthotopic mouse models of PC 2. Compare the molecular effect on prostate cancer and immunological response induced by 161Tb-PSMA and 177Lu-PSMA treatment 3. Imaging of prostate cancer patients to assess 161Tb-PSMA biodistribution and safety. We intend to develop a radiopharmaceutical for prostate cancer (PC) treatment able to specifically target cancer cells at the very early biochemical recurrence, that occurs in a substantial proportion of patients. Indeed, men treated with curative intent for localized prostate cancer have a 27-53% chance of recurrence within 10 years. Thereafter, prognosis is linked to the identification of metastatic spread. For the success of the second-line therapy, it is crucial to treat metastatic foci as early as possible, when metastases are small and undetectable on imaging. Targeted radiopharmaceuticals labelled with optimal radionuclide, instead, can effectively reach disseminated tumor occult micrometastases in multiple body locations and deliver cell-killing radiation. We intend to translate 161Tb-PSMA into clinics and provide prostate cancer patients with an effective treatment option.

Dose verification after 225Ac-Targeted Alpha Therapy (TAT) remains challenging because of the low probability of gamma co-emissions and the overlapping Bremsstrahlung of beta-emitters in the 225Ac decay chain. Recent studies have reported on quantitative 225Ac-SPECT imaging of patients undergoing 225Ac-TAT by mainly using the gamma emissions at 440 keV from its daughter 213Bi. However, daughter isotopes of 225Ac don't remain conjugated to the molecular carrier because of the emission of an α-particle and the recoiling daughter effect. Therefore, daughter radionuclides can redistribute, and the predicted cumulated activities for 225Ac based on gamma emissions by daughter radionuclides can be biased such that dosimetry calculations should account for potential differences in biodistribution between 225Ac and especially 213Bi. To anticipate clinical studies with 225Ac-TAT, this project will use dedicated phantom measurements to evaluate and select the optimal imaging protocols for quantitative 225Ac-(micro)SPECT using state-of-the-art imaging systems. In addition, it will determine whether a redistribution of 213Bi can be anticipated and differences between 225Ac and 213Bi activity concentrations can be quantified. To anticipate the analysis of blood samples of patients undergoing 225Ac-TAT, this will be combined with optimal gamma counter measurement protocols to quantify different 213Bi /225Ac activity ratios in solutions. Finally, a new spectral SPECT image reconstruction approach using both the 221Fr and 213Bi photopeak window will be developed and compared to the current state of the art 225Ac-SPECT reconstruction in terms of quantitative accuracy and image quality.

The yield of 211At is currently limited by the restriction the incident α-beam energy of the 209Bi(α, 2n)211At reaction to 28 MeV avoiding the co-production of 210At with its daughter 210Po through 209Bi(α, 3n)210At. In addition, 210Po is produced as well through the direct 209Bi(α, x)210Po reaction at a threshold of 26.7 MeV. Cross-sectional data predict however a significant increase of the 211At-yield at higher energies indicating that this approach deserves serious consideration. The impact of toxic 210Po in application of TAT using 211At‐labelled sdAbs cannot be based on the extrapolation of theoretical or even experimentally measured activities of 210At at end of bombardment (EOB). It requires a detailed characterisation of activity balances as a function of the production method during all specific steps towards the clinical application and even beyond. Activity balances of At- and Po-radionuclides during production, target dissolution, separation by extraction chromatography and labelling will give detailed insight in gaseous discharges and liquid/solid waste production. Ex vivo quantification of At- and Po-radionuclides in mice treated with 211At‐labelled sdAbs will enable to predict patient toxicity by assessing the contribution of 210Po to the absorbed dose in tumours and healthy tissues. The detailed pharmacokinetic and toxicological data will also be the basis of risk analysis during the application of 211At‐labelled sdAbs in a clinical setting. FIAPo will assist PRISMAP by proposing solutions to tackle an issue related to the problem of the availability of 211At. The impact of increased incident alpha beam energy to increase the yield of 211At will be fully studied. Not only the impact on dosimetry, but also the impact on fields such as radioactive waste management and enhanced nuclear safety measures will be investigated. In this way FIAPo will contribute the future clinical translation of TAT with 211At‐labelled sdAbs.

Radiation therapy, in which ionizing radiation is administered locally to the tumor via an external beam or by surgical implantation of radionuclide-based seeds, is one of the gold standard treatments for cancer. Due to the non-selective nature of radiation, healthy tissue surrounding the cancerous region is often affected by the treatment. Therefore, new strategies are being studied to improve the selectivity of the treatment and minimize side effects. However, several challenges limit the current development of targeted radiotherapy, such as functionalization of the therapeutic agent (radioactive isotope) with vectors to enhance its accumulation in target tissue and control of release. Nanoparticles offer unique opportunities as treatment delivery vehicles, since they have a large surface area that allows incorporation of a high amount of therapy (drug or isotope), improve cellular uptake of drugs and are easily functionalized with biomolecules for further accumulation in target processes. In the development of new nanotechnological tools, a new area of research has begun to emerge based on natural exosomes. Exosomes are small, 30-140 nm, membrane-defined particles of endosomal origin. Their natural origin provides them with greater biocompatibility and lower immuno-responsiveness, thus emerging as new nanotechnological tools, not only in diagnostics but also as platforms for the controlled release of drugs. In addition and due to their natural migration in tumor tissues and pre-metastasis niche, numerous current studies have begun to evaluate the role of exosomesas non-toxic and biocompatible nanoplatforms for drug delivery. The main objective of this work is the development of new radiotheragnostic agents based on natural nanoparticles (exosomes) radioactively labeled with the novel therapeutic and diagnostic isotope Terbium 161 (161Tb).

Fibroblast activation protein (FAP) is a serine protease expressed on stromal cells most of epithelial cancers, whereas its expression is almost undetected in normal tissues. In addition, FAP expression is highly restricted and transiently increased in adult tissues during wound healing, inflammation or fibrosis in activated fibroblasts. Among the stromal cells, cancer associated fibroblasts (CAFs) having a FAP-positive phenotype have been associated with poor prognosis in multiple cancers. The highly focal expression and cancer-specific distribution of FAP make this protein a promising cancer diagnostic marker and an attractive therapeutic target. Motivated by the success of FAP-targeted positron emission tomography (PET) radiotracers, FAP-targeted radionuclide therapies are currently heavily investigated. In addition, FAP-targeted radiopharmaceuticals offer the possibility of imaging diagnostics and targeted radionuclide therapy using the same ligand (theranostics), enabling personalized cancer treatment. However, the relatively rapid washout from the tumor and inadequate pharmacokinetics (PK) of current FAP ligands represents a major problem for radioligand therapy. Therefore, the goal of this application is to prolong the tumor residence and improve PK making FAP ligands into efficient radiotherapeutics. We will use a pretargeting strategy to improve the tumor targeting, and at the same time avoid the high blood residence of FAP radiotracers. We will develop radiolabeled trans-cyclooctenes (TCOs) containing the 18F/211At theranostic pair, and dimeric FAP ligands containing a tetrazine moiety for pretargeted radiotheranostics. Radiotracers will be evaluated in vitro to assess FAP activity and selectivity. Finally, a human pancreatic cancer mouse model will be used to assess both imaging and therapeutic potential of our FAP radiotracers. If successful, our strategy will help physicians select patients who can benefit from FAP-targeted radionuclide therapy.

Fibroblast activation protein inhibitors (FAPi) are a class of molecules that have shown promising results in the diagnosis and therapy of cancer and several other diseases. FAP is a protein target overexpressed by fibroblasts in diseased sites, including tumour microenvironment, which is not expressed in healthy tissue, whose expression seems to correlate with tumour aggressiveness. 68Ga-labelled FAPi ligands have been extensively tested in preclinical and clinical models. The majority of the developed molecules take advantage of DOTA-like chelators for an efficient and stable 68Ga labeling. However, the ligand AAZTA has recently shown interestingly properties for nuclear medicine application. Indeed, while DOTA chelation required harsh labeling conditions, AAZTA required 10 minutes at room temperature for efficient and stable chelation. Recently, some research groups demonstrated the advantages of using AAZTA over DOTA chelators to develop radioactive FAPi molecules. This proposal aims at producing and preclinical testing a theranostic pair based on the AAZTA-FAPi-46 ligand radiolabelled with 152Tb/149Tb isotopes. 152Tb is a positron emitter with a half-life of 17.2 h with clinical potential for performing patient-specific PET dosimetry before targeted radiotherapy, and it can be easily coupled with 149Tb, a nuclide with a half-life of 4.12 h able to co-emit short range high energy alfa-particles and positrons for simultaneous radiotherapy and imaging. Though the complexation of Tb(III) ions in the AAZTA cage has not been reported yet, the published data on the coordination of its two neighbours within the lanthanide series, Gd(III) and Dy(III), provide a strong indication that Tb-AAZTA will keep the favourable coordination properties displayed by Gd(III), Lu(III), Ga(III), and Sc(III) chelates. This project is therefore characterized by a high grade of innovation but keeping a good level of feasibility.

This project aims at exploring alternatives of production of 165Er, which was recently used in bimodal imaging SPECT/MRI for Zn(II) quantification. We have recently produced for the first time in France 165Er by proton/deuton irradiation. We are now exploring novel ways of production or alternatives to 165Er: (1) production of 165Tm by spallation and creation of a generator 165Tm/165Er (2) Use of 155Tb as an alternative to 165Er for SPECT. This project will focus on the development and optimization of the 165Tm/165Er generator particularly by using innovative grafted materials for Ho/Er/Tm separation and purification. For imaging applications, the radiolabeling yield is of prime importance, therefore we will study the complexation with various Zn(II) sensitive ligands. Finally, we aim to use 155Tb as a surrogate for 165Er in a preclinical setting, bearing in mind the potential clinical translation of our novel imaging probes.

The Laboratoire Radiopharmaceutiques Biocliniques (UMR S1039) in Grenoble, France, has recently developed A1, a nuclear imaging agent targeting Mesothelin, a 40kDa GPI-anchored membrane protein which expression is limited in healthy tissues but that is overexpressed in nearly 40% of solid tumors, such as pancreatic cancer, triple negative breast cancer and ovarian cancer. The objective of the present project is to engineer A1 so that it can be radiolabeled with the therapeutic radioisotopes Lu-177 and Tb-161, and to evaluate their potency in vitro and in vivo in mice bearing human breast or pancreatic tumours.

A comparative study on 161Tb- and 177Lu-labeled somatostatin receptor agonists and antagonists recently published, revealed an improved therapeutic efficacy of the latter when 161Tb-labeled due to a suspected, increased damage to the cell membrane. Similar outcomes are thus assumed for other transmembrane receptor antagonists, such as those targeting the gastrin-releasing peptide receptor (GRPR). Therefore, a comparative study on two GRPR antagonists, RM2 (DOTA-Pip5-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) and the recently introduced, metabolically more stable AMTG (DOTA-Pip5-D-Phe6-Gln7-α-Me-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2), will be carried out with 161Tb and the commonly applied 177Lu. Due to the assumption that more severe damage will be caused to the cell membrane if a high percentage of the radiolabeled peptide is receptor-bound but not internalized, these values will be determined. Furthermore, biodistribution studies and µSPECT/CT imaging will be performed at 1, 4, 24 and 72 h post-injection in PC-3 tumor-bearing mice to investigate pharmacokinetics of RM2 and AMTG with both 161Tb and 177Lu over time. An added value of 161Tb over 177Lu will be examined via a therapy study in PC-3 tumor-bearing mice. Thereby, an improved therapeutic efficacy is anticipated for 161Tb-AMTG and 161Tb-RM2 over their 177Lu-labeled analogues, as already observed for 161Tb-labeled somatostatin receptor antagonists. Moreover, it will be interesting to see whether the improved metabolic stability of the AMTG peptide further adds some benefit to the therapeutic efficacy, particularly when 161Tb-labeled. Based on previous data on 177Lu-AMTG and the encouraging results of 161Tb-labeled somatostatin receptor antagonists, an even improved therapeutic efficacy using 161Tb-AMTG is conceivable. 161Tb-AMTG might thus become a clinically applied compound for targeted radiotherapy of GRPR-expressing malignancies, such as prostate and breast cancer, in the near future.