PRISMAP funded projects

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.