Few PET isotopes are suitable for antibody labelling since immunoPET requires that the PET isotope can be attached to the mAb with high in-vivo stability and the decay half-life of the isotope should match the pharmacokinetics of the mAb (Phelps 2004). Both 124I (t½ = 4.2 d) and 89Zr (t½ = 3.3 d) have a near ideal half-life for anti-body-based imaging, but there are several ad-vantages of using 89Zr over 124I. For 124I, the high energy of its positrons (2.13 MeV), results in a relatively low PET image resolution and the possible dehalogenation in vivo can lead to significant radioactivity uptake in non-targeted organs. In comparison, for 89Zr the low energy of its positron (395.5 keV), results in a PET images with a higher spatial resolution and furthermore, 89Zr is a residualizing isotope, which is trapped inside the target cell after internalization of the mAb. One disadvantage of 89Zr is its abundant high energy gamma-ray (909 keV), which may limit the radioactive dose that can be administered to the patients. The most popular reaction to produce 89Zr is the 89Y(p,n)89Zr nuclear reaction (Sahar et al., 1966; Link et al., 1986). A proton beam with 14-16MeV energy is used to bombard inexpensive high-purity 89Y metal target (99.9%), avoiding cumbersome recycling of the target material. The yttrium targets could be either a foil (Dejesus and Nickels, 1990), sputtered onto a copper support (Meijs et al., 1994) or Y2O3 pellets (S. A. Kandil, B. Scholten, 2007). Although 89Zr is currently commercially available, its price is prohibitive for routine clinical applications of 89Zr immuno-PET. The motivation of the present work was the fully automated production of small quantities of 89Zr using commercially available automated systems. We also describe a newly designed and tested platinum cradle, capable of holding a metallic foil and being directly transferable/compatible between the IBA NIRTA target and IBA Pinctada Metal dissolution/purification module. Material and Methods The solid target infrastructure used for 89Zr production was identical to the implementation reported earlier for production of 64Cu and 124I (S. Poniger et al. 2012). The commercially avail-able Nirta Solid Target from IBA was coupled to our 18/9 IBA cyclotron using a 2-meter external beam line. A fully automated pneumatic solid target transfer system (STTS) designed by TEMA Sinergie was used to deliver the irradiated tar-gets to a dedicated hotcell. The newly designed platinum cradle holding the yttrium foil (0.127 mm thick, 8 mm d) is shown in FIG. 1. Typical irradiation parameters were 14.9 MeV at 20 μA for 1.5 hours (90o angle of incidence). The irradiated cradle, containing the 89Zr target is then loaded directly into the IBA Pinctada Metal module (see FIG. 2) for dissolution/purification without disassembly. We used the dissolution/purification method described by Holland et al. 2009, without modification (Purification of 89Zr from 89Y, 88Y and other radionuclidic impurities using a hydroxamate column, with 89Zr eluted with 1.0M Oxalic acid). Radionuclidic purities were evaluated by gamma spectroscopy and traces of metallic impurities were determined by ICP-MS. Results and Conclusion FIGURE 3 shows the gamma spectrum of the purified 89Zr solution. Since yttrium has one stable isotope only, relatively pure 89Zr is produced at low energy (14.9 MeV). In these preliminary non-optimized cyclotron productions, average purified 89Zr yield of 0.34 mCi/μAh was achieved, in comparison to values of 1.5 mCi/μAh found in the literature (10° angle of incidence). In these preliminary experiments, no deformation of the foil was observed at 20 μA beam current and higher currents are under investigation.

Introduction Zr-89 is a useful radionuclide for radiolabeling proteins and other molecules.1,2 There are many reports of cyclotron production of 89Zr by the 89Y (p,n) reaction. Most irradiations use thin metal backed deposits of Y and irradiation currents up to 100 µA or thicker amounts of Y or Y2O3 with ~ 20 µA irradiations.3,4 We are working to develop high specific activity 89Zr using a low energy 11 MeV cyclotron. We have found that target Y metal contains carrier Zr and higher specific activities are achieved with less Y. The goal of this work was to optimize yield while minimizing the amount of Y that was irradiated. Material and Methods All irradiations were done using a Siemens Eclipse 11 MeV proton cyclotron. Y foils were used for the experiments described here. Y2O3 was tried and abandoned due to lower yield and poor heat transfer. Yttrium metal foils from Alfa Aesar, ESPI Metals and Sigma Aldrich, 0.1 to 1 mm in thickness, were tested. Each foil was irradiated for 10 to 15 minutes. The targets to hold the Y foils were made of aluminum and were designed to fit within the “paper burn” unit of the Siemen’s Eclipse target station, allowing the Y target body to be easily inserted and removed from the system. Several Al targets of 2 cm diam. and 7.6 cm long were tested with the face of the targets from 11, 26 or 90o relative to the beam to vary watts cm−2 on the foil. The front of the foils was cooled by He convection and the foil backs by conduction to the Al target body. The target body was cooled by conduction to the water cooled Al sleeve of the target holder. Results and Conclusion The best target was two stacked, 0.25 mm thick, foils to stop beam. 92% of the 89Zr activity was in the front 0.25 mm Y foil. With the greatest slant we could irradiate up to 30 µA of beam on tar-get. However, the 13×30 mm dimensions of the foil was more mass (0.41 g) and lower specific activity than was desired. Redesign of the target gave a target 90o to the beam with 12×12 mm foils (0.15 g/foil) that were undamaged with up to 30 µA irradiation when two foils were used. This design has a reduction in beam at the edges of ~10%. With this design, a single Y foil, 0.25 mm thick sustained over 31 µA of beam and a peak power on target of 270 watts cm−2. The product was radionuclidically pure 89Zr after all 89mZr and small amounts of 13N produced from oxygen at the surface had decayed (TABLE 1). Our conclusion is that the optimum target is a single 0.25 mm thick Y foil to obtain the greatest specific activity at this proton energy. This produces 167 MBq of 89Zr at EOB with a 15 minute and 31 µA irradiation. We are continuing to redesign the clamp design to reduce losses at the edge of the beam.

Few PET isotopes are suitable for antibody labelling since immunoPET requires that the PET isotope can be attached to the mAb with high in-vivo stability and the decay half-life of the isotope should match the pharmacokinetics of the mAb (Phelps 2004). Both 124I (t½ = 4.2 d) and 89Zr (t½ = 3.3 d) have a near ideal half-life for anti-body-based imaging, but there are several ad-vantages of using 89Zr over 124I. For 124I, the high energy of its positrons (2.13 MeV), results in a relatively low PET image resolution and the possible dehalogenation in vivo can lead to significant radioactivity uptake in non-targeted organs. In comparison, for 89Zr the low energy of its positron (395.5 keV), results in a PET images with a higher spatial resolution and furthermore, 89Zr is a residualizing isotope, which is trapped inside the target cell after internalization of the mAb. One disadvantage of 89Zr is its abundant high energy gamma-ray (909 keV), which may limit the radioactive dose that can be administered to the patients. The most popular reaction to produce 89Zr is the 89Y(p,n)89Zr nuclear reaction (Sahar et al., 1966; Link et al., 1986). A proton beam with 14-16MeV energy is used to bombard inexpensive high-purity 89Y metal target (99.9%), avoiding cumbersome recycling of the target material. The yttrium targets could be either a foil (Dejesus and Nickels, 1990), sputtered onto a copper support (Meijs et al., 1994) or Y2O3 pellets (S. A. Kandil, B. Scholten, 2007). Although 89Zr is currently commercially available, its price is prohibitive for routine clinical applications of 89Zr immuno-PET. The motivation of the present work was the fully automated production of small quantities of 89Zr using commercially available automated systems. We also describe a newly designed and tested platinum cradle, capable of holding a metallic foil and being directly transferable/compatible between the IBA NIRTA target and IBA Pinctada Metal dissolution/purification module. Material and Methods The solid target infrastructure used for 89Zr production was identical to the implementation reported earlier for production of 64Cu and 124I (S. Poniger et al. 2012). The commercially avail-able Nirta Solid Target from IBA was coupled to our 18/9 IBA cyclotron using a 2-meter external beam line. A fully automated pneumatic solid target transfer system (STTS) designed by TEMA Sinergie was used to deliver the irradiated tar-gets to a dedicated hotcell. The newly designed platinum cradle holding the yttrium foil (0.127 mm thick, 8 mm d) is shown in FIG. 1. Typical irradiation parameters were 14.9 MeV at 20 μA for 1.5 hours (90o angle of incidence). The irradiated cradle, containing the 89Zr target is then loaded directly into the IBA Pinctada Metal module (see FIG. 2) for dissolution/purification without disassembly. We used the dissolution/purification method described by Holland et al. 2009, without modification (Purification of 89Zr from 89Y, 88Y and other radionuclidic impurities using a hydroxamate column, with 89Zr eluted with 1.0M Oxalic acid). Radionuclidic purities were evaluated by gamma spectroscopy and traces of metallic impurities were determined by ICP-MS. Results and Conclusion FIGURE 3 shows the gamma spectrum of the purified 89Zr solution. Since yttrium has one stable isotope only, relatively pure 89Zr is produced at low energy (14.9 MeV). In these preliminary non-optimized cyclotron productions, average purified 89Zr yield of 0.34 mCi/μAh was achieved, in comparison to values of 1.5 mCi/μAh found in the literature (10° angle of incidence). In these preliminary experiments, no deformation of the foil was observed at 20 μA beam current and higher currents are under investigation.

Notre travail s’est focalisé sur l’utilisation du trastuzumab‐immunoPET afin d’étudier et deguider une approche nouvelle visant à surmonter la résistance au médicament trastuzumab,causée par la surexpression de MUC4 dans le cancer du sein.Pour ce faire, nous avons préparé et utilisé du 89Zr‐trastuzumab dans le but de suivresa capacité de liaison au récepteur HER2 ainsi que son accumulation dans des cellulescancéreuses mammaires. Ensuite, nous avons formulé l’hypothèse que des agentsmucolytiques, tels que la N‐Acétylcystéine (NAC), en démêlant les réseaux formés par lesmucines, permettent l’amélioration de la captation du radiotraceur in vitro et in vivo. Eneffet, l’addition du NAC a occasionné une accumulation significative de 89Zr‐trastuzumab,sans altération ni changement de l’affinité de liaison au récepteur. Ceci semble égalementproduire une meilleure sensibilité des imageries PET dans le modèle animal choisi.Dans une seconde étape, nous avons évalué, dans un modèle murin de cancer du seinrésistant au trastuzumab et surexprimant la MUC4, si cette captation accrue se traduit parun bénéfice thérapeutique en utilisant le NAC combiné au trastuzumab. Nous avons obtenuun effet inhibiteur qui réduit de moitié la croissance tumorale, comparable à celui observépour la tumeur mammaire sensible au trastuzumab (implantée dans le même animal).En conclusion, notre étude démontre l’efficacité de l’utilisation de traceurs PETsurtout à visée théranostique, comme c’est le cas du 89Zr‐trastuzumab, pour étudier etévaluer la résistance aux médicaments ciblés apparentés au radiotraceur lui‐même. Ellepropose l’utilisation du NAC pour améliorer l’accessibilité du récepteur pour le radiotraceurainsi que pour le médicament « froid » ouvrant, de ce fait, une perspective vers uneutilisation clinique chez un sous‐type de patientes atteintes d’un cancer du sein. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

The aim of this thesis is to synthesize new kind of macrocyclic ligands for complexation of Zr4+ . Zirconium(IV) complexes have potential as contrast agents in immuno-PET. Three macrocyclic ligands L1, L2 and L3 with dif- ferent pendant arms (HOPO, maltol, hydroxamate) were designed. Despite numerous attempts syntheses of L1 and L2 were unsuccessful. Ligand L3 was synthesized and the protonation constants were determined by poten- tiometric titration. Complexes [Zr4+ (L3)] were studied too. Catechol ligand L4 was prepared and complexes with cations Zn2+ , Ga3+ and Zr4+ were investigated. 1

Introduction Zr-89 is a useful radionuclide for radiolabeling proteins and other molecules.1,2 There are many reports of cyclotron production of 89Zr by the 89Y (p,n) reaction. Most irradiations use thin metal backed deposits of Y and irradiation currents up to 100 µA or thicker amounts of Y or Y2O3 with ~ 20 µA irradiations.3,4 We are working to develop high specific activity 89Zr using a low energy 11 MeV cyclotron. We have found that target Y metal contains carrier Zr and higher specific activities are achieved with less Y. The goal of this work was to optimize yield while minimizing the amount of Y that was irradiated. Material and Methods All irradiations were done using a Siemens Eclipse 11 MeV proton cyclotron. Y foils were used for the experiments described here. Y2O3 was tried and abandoned due to lower yield and poor heat transfer. Yttrium metal foils from Alfa Aesar, ESPI Metals and Sigma Aldrich, 0.1 to 1 mm in thickness, were tested. Each foil was irradiated for 10 to 15 minutes. The targets to hold the Y foils were made of aluminum and were designed to fit within the “paper burn” unit of the Siemen’s Eclipse target station, allowing the Y target body to be easily inserted and removed from the system. Several Al targets of 2 cm diam. and 7.6 cm long were tested with the face of the targets from 11, 26 or 90o relative to the beam to vary watts cm−2 on the foil. The front of the foils was cooled by He convection and the foil backs by conduction to the Al target body. The target body was cooled by conduction to the water cooled Al sleeve of the target holder. Results and Conclusion The best target was two stacked, 0.25 mm thick, foils to stop beam. 92% of the 89Zr activity was in the front 0.25 mm Y foil. With the greatest slant we could irradiate up to 30 µA of beam on tar-get. However, the 13×30 mm dimensions of the foil was more mass (0.41 g) and lower specific activity than was desired. Redesign of the target gave a target 90o to the beam with 12×12 mm foils (0.15 g/foil) that were undamaged with up to 30 µA irradiation when two foils were used. This design has a reduction in beam at the edges of ~10%. With this design, a single Y foil, 0.25 mm thick sustained over 31 µA of beam and a peak power on target of 270 watts cm−2. The product was radionuclidically pure 89Zr after all 89mZr and small amounts of 13N produced from oxygen at the surface had decayed (TABLE 1). Our conclusion is that the optimum target is a single 0.25 mm thick Y foil to obtain the greatest specific activity at this proton energy. This produces 167 MBq of 89Zr at EOB with a 15 minute and 31 µA irradiation. We are continuing to redesign the clamp design to reduce losses at the edge of the beam.