International Incoming Fellowships (IIF) Call: FP7-People-2011-IIF

Transcription

1 PEOPLE MARIE CURIE ACTIONS International Incoming Fellowships (IIF) Call: FP7-People-2011-IIF Final report: Radiopharm Metal Iso (299009)

2 Publishable summary (i) New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with SSTR2- and αvβ3-targeting peptides, and an immunoconjugate of the HER2-targeting trastuzumab Objectives: To synthesise and evaluate labelling and biodistribution of conjugates of new isothiocyanatefunctionalised tris(hydroxypyridinone) chelators with tumour targeting peptides. Methods: Two tris(hydroxypyridinones) each with a pendant isothiocyanate group were synthesised and conjugated to amine groups of Tyr 3 -octreotate for SSTR2 targeting (1), monovalent and trivalent cyclic-(rgdfk) derivatives (e.g. 2) for α v β 3 targeting (Figure) and the monoclonal antibody trastuzumab. Eluate from an ithemba Labs 1.8 GBq 68 Ge/ 68 Ga generator was processed on a cation-exchange cartridge, eluted in 90% ethanol/0.9 N HCl (J Nucl Med 2014, 55:1023), and added to each bioconjugate before neutralising with ammonium acetate. Biodistribution was assessed in balb/c nude mice bearing U87MG (αvβ3) or AR42J (SSTR2) tumours, by PET at 1 and 2 h post-injection, after which animals were killed and tissues/organs harvested and counted. Co-injection of unconjugated peptide (400 µg per animal) with each radiotracer, followed by scanning and biodistribution 1 h postinjection assessed specificity of the radiotracers. Results: All peptide conjugates were labelled with 68 Ga 3+ in 95% radiochemical yield and high specific activity ( MBq nmol -1 ) at 25 C and formulated to ph 6-7 in < 5 min. Biodistribution showed tumour-specific uptake and retention of target receptor affinity. Excretion was rapid and predominantly renal (Figure). Co-injection of unconjugated peptide substantially reduced tumour uptake in all cases. A HER2-targeting mab, trastuzumab, was radiolabeled in < 5 min by direct addition of 68 Ga 3+ generator eluate to a solution of immunoconjugate at ph 6 7, with specific activities of 50 MBq nmol -1. The radiolabeled immunoconjugate accumulated specifically in HER2-expressing cells in vitro. Significance: The new bifunctional tris(hydroxypyridinone) chelators enable easy preparation of bioconjugates, and rapid radiolabelling with 68 Ga 3+ under mild conditions without subsequent purification. The simplicity and efficiency of labelling at very low concentrations under mild conditions brings, for the first time, the possibility of kit-based 68 Ga tracer production without complex automated synthesis typical of multistep PET radiochemistry. This would greatly increase 68 Ga PET access to hospitals, expanding the use of the 68 Ga generator. (ii) A tripodal tris(hyroxypyrdinone) ligand for immunoconjugate PET imaging with zirconium-89 Objectives: Due to its long half-life (78.4h) and decay properties (β +, 897keV), 89 Zr is an appealing radionuclide for immunopet imaging with antibodies. Desferrioxamine-B (DFO) is the most widely used bifunctional chelator to coordinate 89 Zr 4+ because the radiolabelling of the resulting immunoconjugates is rapid under mild conditions. 89 Zr-DFO complexes are stable in vitro but recent data demonstrated the release and subsequent bone uptake of the

3 radiometal in vivo. We aim to evaluate a tripodal tris(hydroxypyridinone) ligand CP256 (Berry DJ, et al. (2011) ChemComm, 47, ) developed in our laboratory, as a 89 Zr 4+ chelator and compare it with DFO. Methods: To compare the ability of CP256 to complex Zr 4+ with that of DFO, decreasing concentrations of each chelator were labelled with [ 89 Zr(oxalate) 4 ]. Competition experiments were also undertaken. The radiolabelled products and radiochemical yields were evaluated by ITLC and reverse phase HPLC-MS coupled to a scintillation detector. To assess the stability of DFO/CP256-immunoconjugates radiolabeled with 89 Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Serum stability studies and in vivo biodistribution and micropet/ct studies with normal C57Bl/6j mice were undertaken on the 89 Zr-radiolabelled immunoconjugates. Results: CP256 coordinates Zr 4+ (natural abundance) and comparison of HPLC-MS/scintillator chromatograms confirms that addition of [ 89 Zr(oxalate) 4 ] to CP256 results in formation of [ 89 Zr(CP256)] +. DFO and CP256 can be radiolabelled with [ 89 Zr(oxalate) 4 ] at ambient temperature in quantitative yield at ph 6-7 at millimolar concentrations. Competition experiments demonstrate that 89 Zr 4+ does not substantially dissociate (<10%) from [ 89 Zr(CP256)] + in the presence of a ten-fold greater concentration of DFO relative to CP256. However, 89 Zr 4+ dissociates from [ 89 Zr(DFO)] 2+ in the presence of one equivalent of CP256 (relative to DFO), resulting largely in [ 89 Zr(CP256)] +. The immunoconjugates, CP256-trastuzumab and DFO-trastuzumab, can be labelled with 89 Zr in >98% yield at specific activities of 55MBq mg -1 and 91MBq mg -1 respectively. Both 89 Zr-labelled immunoconjugates are stable in serum with respect to dissociation of the radiometal. MicroPET/CT and biodistribution studies indicate that after one day, 89 Zr 4+ dissociates from CP256-trastuzumab with significant amounts of activity associated with bones and joints (25.88±0.58% ID g -1 after one week). In contrast, <8% ID g -1 of 89 Zr activity is associated with bone for animals administered 89 Zr-DFO-trastuzumab over the course of one week. Significance: The tris(hydroxypyridinone) ligand, CP256 coordinates 89 Zr 4+ rapidly under mild conditions, but the 89 Zr-labelled immunoconjugate, 89 Zr-CP256-trastuzumab, was observed to release significant amounts of 89 Zr 4+ in vivo, demonstrating inferior stability when compared with 89 Zr-DFO-trastuzumab. Project objectives The following aims were originally proposed: 1. Synthesise bifunctional/trifunctional chelators for 99m Tc and 188 Re that can be attached to targeting molecules (αmsh-based peptide and bisphosphonate), providing multimeric derivatives for imaging studies. These derivatives will be further developed to provide molecular 99m Tc and 188 Re agents with improved target affinity/tumour uptake. 2. Test the hypothesis that multivalency (rather than prolonged bioavailability of a radiotracer) is the main contributor to enhanced tumour uptake. 3. Synthesise a multivalent chelator platform that can be attached to a targeting molecule for a pre-targeting approach using αmsh/mt1r as a model targeting system so that targeting molecules with slow pharmacokinetics such as anti-melanoma antibodies can be used with short half-life radioisotopes and hence reduce radiation dose to patients. 4. Optimise a selected design for development of each of the above towards clinical application. The following milestones were identified as critical to achieving project objectives: 1. Synthesis of novel bifunctional chelators for generator-produced isotopes 2. Conjugation of bifunctional chelators to targeting peptides, and radiolabeling 3. In vitro uptake and in vivo biodistribution of labelled conjugates

4 As described below (Deviations from Annex) the following milestone/objective was also added to the project: 4. Evaluation of tris(hydroxypyridinone) ligands for radiolabeling and imaging antibodies with 89 Zr 4+ Work progress and achievements Progress towards objectives Progress results and research outcomes critical to work progress and achievements are listed under project milestones, and referenced to project aims (in parentheses). Milestone 1: Synthesis of novel bifunctional chelators for generator-produced isotopes A dithiocarbamate (DTC) ligand derived from carbon disulfide and iminodiacetic acid has been synthesized for coordination of the generator-produced SPECT isotope, 99m Tc. The resulting ligand (DTC-IDA)) contains two acetate groups that in future could be activated and conjugated to primary amines of biological targeting molecues. Reaction of this ligand at room temperature with a solution containing (either) the [ 99m TcN] 2+ or [ 99 TcN] 2+ core results in formation of [ 99m TcN(DTC-IDA) 2 ] 2-, as determined by LC-MS coupled to a radioscintillation detector. Furthermore, the ligand reacts with solutions containing Zn 2+, forming [Zn(DTC-IDA) 2 ] 2-, which undergoes transmetallation with [ 99m TcN] 2+ to form [ 99m TcN(DTC-IDA) 2 ] 2-, providing a second radiochemical synthetic route. The DTC-IDA ligand also reacts at room temperature with [ReNCl 2 (PPh 3 ) 2 ] to form [ReN(DTC-IDA) 2 ] 2-. (Aim 1) Two new bifunctional tris(hydroxypyridinone) (THP) chelators (THP-PhNCS and THP-NCS) designed specifically for rapid labeling with 68 Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. (Aim 3) A bifunctional chelator containing three tris(hydroxypyridinone) groups (an enneakis(hydroxypyridinone)) for coordination of up to three 68 Ga 3+ ions has been synthesised THP 3 -PhNCS. This compound also contains an isothiocyanate group that reacts with primary amines. (Aim 3) Milestone 2: Conjugation of bifunctional chelators to targeting peptides, and radiolabeling Both THP-PhNCS and THP-NCS have been conjugated with the primary amine group of a cyclic integrin targeting peptide, cyclic(rgdfk), providing THP-PhNCS-RGD and THP-NCS-RGD. Each conjugate can be radiolabeled and formulated for intravenous injection by treatment with generator-produced 68 Ga 3+ in over 95 % radiochemical yield under ambient conditions in less than 5 min, with specific activities of MBq nmol -1. Additionally, a scaffold has been prepared from which three peptidic cyclic integrin targeting groups (RGD peptide) for binding the αvβ3 integrin receptor have been attached. Conjugation of the new bifunctional chelators THP-PhNCS and THP 3 -PhNCS with the scaffold containing multiple peptide groups has yielded a trimeric compound that contains three peptidic groups and one chelator group (THP-PhNCS-RGD 3 ), and a dendritic compound that contains three peptidic groups and three chelator groups (THP 3 -PhNCS-RGD 3 ). (Aim 3) THP-NCS has been conjugated to the somatostatin receptor 2 (SSTR2) targeting peptide, Tyr 3 -octreotate (TATE) via a PEG linker to provide THP-NCS-TATE, for comparison with the clinically used conjugate, DOTA- TATE. THP-NCS-TATE can be radiolabeled with 68 Ga 3+ in over 95 % radiochemical yield in specific activities of up to 100 MBq nmol -1. (Aim 3, 4) We are yet to conjugate DTC-IDA to a targeting peptide, but this project will contribute to a continuing programme of research undertaken by the researcher. Milestone 3: In vitro uptake and in vivo biodistribution of 68 Ga-labeled conjugates In vitro measurements and in vivo PET scanning and ex vivo biodistribution in mice bearing α v β 3 -positive U87MG tumours demonstrate that the new 68 Ga 3+ -labeled THP peptide conjugates retain affinity for the α v β 3 - integrin receptor, clear within 1 2 h from circulation predominantly via a renal route and undergo receptormediated tumor uptake in vivo. In vivo tumour uptake and retention of [ 68 Ga(THP-PhNCS-RGD] is higher than that of [ 68 Ga(THP-NCS-RGD]. (Aim 2, 4)

5 The 68 Ga-labelled trimeric conjugate, [ 68 Ga(THP-PhNCS-RGD 3 )] demonstrates higher tumour accumulation and a higher tumour to blood ratio at 1 and 2 h post-injection in vivo than monomeric [ 68 Ga(THP-PhNCS-RGD)]. Indeed, blood activity at 1 h PI for both conjugates is the same (a measure of similar bioavailability), but tumor uptake of the trimeric conjugate (4.28±0.53 %ID g -1 ) is significantly higher than that of the monomeric compound (2.86±0.43 %ID g -1 ). (Aim 2, 4) Differences in accumulated radioactivity in tumours for trimeric [ 68 Ga(THP-PhNCS-RGD 3 )] and dendritic [( 68 Ga) n (THP 3 -PhNCS-RGD 3 )] (n=1-3) are not statistically significant. Notably, high liver uptake of dendritic [( 68 Ga) n (THP 3 -PhNCS-RGD 3 )] renders this specific tracer entirely unsuitable for PET imaging, however in vivo experiments demonstrate that attachment of a bulky enneakis(hydroxypyridinone) does not adversely decrease the affinity of a peptide targeting group for tumour receptors in vivo. (Aim 2, 4) In vitro experiments have shown that the 68 Ga-labeled radiotracer, [ 68 Ga(THP-NCS-TATE)] specifically binds AR42J receptors and is internalised rapidly, consistent with reported agonist behaviour of octreotate peptide derivatives. In vivo PET scanning and ex vivo biodistribution studies demonstrate that tumour uptake of [ 68 Ga(THP- NCS-TATE)] is comparable to the clinically used radiopharmaceutical, [ 68 Ga(DOTATATE)] in mice bearing SSTR2-positive AR42J tumours. However, in comparison to [ 68 Ga(DOTATATE)], [ 68 Ga(THP-NCS-TATE)] has prolonged kidney retention. (Aim 4) Milestone 4: Evaluation of tris(hydroxypyridinone) ligands for radiolabeling and imaging antibodies with 89 Zr 4+ The tris(hydroxypyridinone) chelator, H 3 CP256 and its bifunctional maleimide derivative, H 3 YM103, was evaluated for coordination of Zr 4+. The NMR spectra, and the 89 Zr 4+ radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabeled immunoconjugates was compared with those of the commercial standard used for 89 Zr 4+ coordination, H 3 DFO and its analogs. H 3 CP256 coordinates 89 Zr 4+ at carrier-free concentrations forming [ 89 Zr(CP256)] +. Competition experiments demonstrate that 89 Zr 4+ dissociates from [ 89 Zr(DFO)] + in the presence of one equivalent of H 3 CP256 (relative to H 3 DFO) at ph 6 7, resulting largely in [ 89 Zr(CP256)] +. To assess the stability of H 3 DFO and H 3 YM103 immunoconjugates radiolabeled with 89 Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labeled with 89 Zr 4+ in >98 % yield at high specific activities and the labeled immunoconjugates were stable in serum with respect to dissociation of the radiometal. In vivo studies in mice indicate that 89 Zr 4+ dissociates from YM103-trastuzumab with significant amounts of activity becoming associated with bones and joints (25.88 ± 0.58 % ID g -1 7 days post-injection). In contrast, < 8 % ID g -1 of 89 Zr activity becomes associated with bone in animals administered 89 Zr-DFO-trastuzumab over the course of 7 days. The significantly lower in vivo stability of the tris(hydroxypyridinone) conjugate is likely to be a result of lower kinetic stability of the Zr 4+ tris(hydroxypyridinone complex) relative to that of DFO and its derivatives. Transfer of knowledge activities Conference and workshop presentations Transfer of knowledge activities such as participation in conferences and workshops are detailed in the section entitled Dissemination Activities, including oral presentations at three conferences. Additionally, the researcher was invited to speak at the workshop entitled Radioisotopes in Cancer Imaging and Therapy (February 2015, London, UK), hosted by the Institute of Cancer Research. Recently, the researcher also represented the Chemistry Cluster/Scientific Programme at the CRUK s KCL/UCL (King s College London/University College London) Comprehensive Cancer Imaging Centre annual meeting (February 2015, London, UK), attended by all programme leads and members of the KCL/UCL CCIC. Student supervision The researcher has supervised four masters-level projects that included: Synthesis of a simple tris(hydroxypyridinone) ligand, and conjugation of the ligand to an antibody and radiolabelling for the resulting conjugate. Radiosynthesis of simple 64 Cu lipophilic compounds based on bis(phosphines). Radiolabelling of peptide-based ligands engineered to coordinate metal ions.

6 In doing so, the fellow trained students to design and synthesis chelators for coordination of radiometals, characterise the structures of relevant metal complexes spectroscopically, evaluate the radiolabeling, and characterise the resulting radiolabelled species. The researcher has also assisted in supervising and training PhD students within the department in areas of design and synthesis of chelators and peptides, radiolabeling of conjugates and evaluation of new radiotracers. Participation in Departmental scientific meetings The researcher has regularly presented laboratory work updates and presentations to her host department, the Department of Imaging Chemistry and Biology within the Division of Imaging Sciences and Bioengineering. In addition to this, the fellow has also presented at Chemistry and Biology meetings where members of the Department of Chemistry, School of Pharmacy and Department of Imaging Chemistry and Biology meet to discuss their research outcomes. Lastly, the fellow has also participated in organising, planning and presenting at monthly meetings of members of the KCL/UCL CCIC chemistry cluster/programme. Collaborations with industry and clinicians The host institute has licensed new tris(hydroxypyridinone) technology to industry partners, and the researcher has liaised with industry partners, CheMatech, providing advice on synthetic procedures to produce commercial tris(hydroxypyridinone) chelator products. These are now available commercially: The researcher was also awarded a Royal Society of Chemistry (RSC) mobility fellowship, allowing her to undertake collaborative research at the Peter MacCallum Centre in Australia, assessing the biology of conjugates synthesised at the host institute in a highly translational research centre/hospital. During this time, the researcher collaborated with radiochemists, biologists and clinicians. Significant results Significant scientific results are outlined in the Publishable summary, and elaborated upon under Progress towards objectives. In brief: A tris(hydroxypyridinone) chelator, CP256, coordinates 89 Zr 4+ rapidly, and immunoconjugates can be radiolabeled in comparable radiochemical yields to that of desferrioxamine immunoconjugates. However, in vivo, 89 Zr 4+ complexes of this tris(hydroxypyridinone) are not stable, resulting in demetallation and accumulation of 89 Zr 4+ in the skeleton. Two new bifunctional tris(hydroxypyridinone) chelators designed specifically for rapid labeling with 68 Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, cyclic(rgdfk). Each conjugate can be radiolabeled by treatment with generator-produced 68 Ga 3+ in high radiochemical yield (> 95 %) under ambient conditions in < 5 min, with specific activities of MBq nmol -1. Biodistribution of an α v β 3 -targeted conjugate showed tumor-specific uptake, retention of target receptor affinity and rapid clearance. The ability to synthesise 68 Ga-based radiopharmaceuticals under mild concentrations at very low concentrations of conjugate brings about the possibility of kit-based 68 Ga radiopharmaceutical production similar to that utilised for 99m Tc-based radiopharmaceuticals that are prevalent in clinical nuclear imaging. This would greatly increase 68 Ga PET access to hospitals, expanding the use of the 68 Ga generator. The novel 68 Ga-labelled SSTR2-targeting conjugate, [ 68 Ga(THP-NCS-TATE)] can be similarly labelled in high radiochemical yield (> 95%) under ambient conditions in < 5 min, with specific activities of up to 100 MBq nmol -1. The radiotracer demonstrates receptor-mediated uptake in AR42J tumours, comparable to that of the clinically-used neuroendocrine molecular imaging agent, [ 68 Ga(DOTATE)], although kidney retention is prolonged in the former radiotracer. A novel trimeric scaffold for conjugation of three peptidic groups has been synthesised, alongside an enneakis(hydroxypyridinone) bifunctional chelator. A novel trimeric αvβ3-targeting cyclic(rgd) conjugate, [ 68 Ga(THP-PhNCS-RGD 3 )] of a tris(hydroxypyridinone) demonstrates higher tumour uptake and higher tumour to background ratios than that of the monomeric conjugate. A trimeric αvβ3-targeting cyclic(rgd) conjugate of the enneakis(hydroxypyridinone) bifunctional chelator has also been prepared, providing the dendritic 68 Ga-labelled radiotracer, [ 68 Ga n (THP 3 -PhNCS-RGD 3 )] (n = 1-3), that can be radiolabeled in very high specific activities (180

7 240 MBq nmol -1 ). The latter dendritic radiotracer demonstrates similar tumour uptake to that of the trimeric radiotracer, although liver uptake substantially reduces the ability of tumours to be visualized clearly by PET scans. Deviations from Annex The researcher has not used the MT1R-targeting αmsh peptide originally specified in the project proposal. Instead, the researcher has substituted this peptide for an αvβ3-targeting cyclic(rgdfk) peptide. This is because (i) expertise and resources were readily available for in vitro and in vivo αvβ3-expressing models, and (ii) it was more propitious to undertake proof-of-concept studies for the well-understood αvβ3 integrin receptor. Additionally, although not originally described in the grant proposal, the researcher has also worked on a related project. This related project uses tris(hydroxypyridinone) ligands (described above) to coordinate the positronemitting isotope, 89 Zr 4+. Since award of the fellowship, this isotope has recently (2012/2013) become commercially available in the European area, and is a significant new technology for PET-based molecular imaging. It was determined that research on this isotope in the context of tris(hydroxypyridinone) ligands would enhance the impact of the research. Use of resources There has been no deviation from planned researcher months. The full budget allocated for laboratory consumables/expenses and travel has been spent. The monies have been spent on laboratory consumables, chemicals, instrument access fees, costs associated with in vivo work, conference registration and associated travel. Dissemination activities As indicated in the publishable summary, the research undertaken over the course of the last two years can be categorised into two separate projects, and thus dissemination activities are reported accordingly. (i) New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with SSTR2- and αvβ3-targeting peptides Two research papers on the tris(hydroxypyridinone) work have been drafted, and the manuscripts are undergoing final review by all authors prior to submission of the manuscript. A third manuscript is currently under preparation. Copies of these drafts can be provided upon request. The tris(hydroxypyridinone) work has recently been presented as an oral presentation at the following conferences: o New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with SSTR2- and αvβ3-targeting peptides, Third Theranostics World Congress on Ga-68 and PRRT, March 2015, Baltimore, USA o Rapid 68 Ga-Radiolabelling of Proteins in Mild Conditions Exploiting a Novel tris(hydroxypyridinone) Bifunctional Chelator, Third Theranostics World Congress on Ga-68 and PRRT, March 2015, Baltimore, USA o One-step, kit-based biomolecule labelling and molecular imaging with gallium-68 tris(hydroxypyridinone) chelators, Radioisotopes in Cancer Imaging and Therapy, February 2015, Institute of Cancer Research, London, UK invited presentation The tris(hydroxypyridinone) work lead to the basis of two successful early career researcher awards: o STEM Early Career Award - NIHR Biomedical Research Centre at Guy s and St Thomas NHS Foundation Trust and King s College London ( 6,972, January March 2015) o Royal Society of Chemistry Research Mobility Fellowship ( 5000, 2014) (ii) A tripodal tris(hyroxypyrdinone) ligand for immunoconjugate PET imaging with zirconium-89 The 89 Zr tris(hydroxypyridinone) work has been presented as an oral presentation at a Dalton Discussions meeting (York, UK; September, 2014) and has recently been published: o Michelle T. Ma*, Levente K. Meszaros, Brett M. Paterson, David J. Berry, Maggie S. Cooper, Yongmin Ma, Robert C. Hider, Philip J. Blower, Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with 89 Zr 4+ : comparison with desferrioxamine-b, Dalton Transactions, 2015, 44,

8 The work has also been presented as an oral presentation at the European Association of Nuclear Medicine Congress, where it was shortlisted for the EANM Marie Curie Award: o Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with 89 Zr 4+ : comparison with desferrioxamine-b, European Association of Nuclear Medicine Congress, October 2013, Lyon, France The work was also presented as a poster at the International Conference on BioInorganic Chemistry 16 (July 2013, Grenoble, France). Project Management Project planning and management has been minimal. Monthly meetings between the scientist in charge (Prof. Blower) and the research fellow (Michelle Ma) have taken place in order to discuss results and plan experiments and reporting. There have been no major changes to planned milestones. Minor deviations from the project proposal are described above, under "work progress and achievements during the period". There have been no gender or ethical issues, and no changes to the legal status of any of the beneficiaries.