Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 Chemical aspects of metal ion chelation in the synthesis and application antibody-based radiotracers Boros, Eszter ; Holland, Jason P Abstract: Radiometals are becoming increasingly accessible and are utilized frequently in the design of ra- diotracers for imaging and therapy. Nuclear properties ranging from the emission of ฀‐rays and ฀+‐particles (imaging) to Auger electron and ฀− and ฀‐particles (therapy) in combination with long half‐lives are ide- ally matched with the relatively long biological half‐life of monoclonal antibodies in vivo. Radiometal labeling of antibodies requires the incorporation of a metal chelate onto the monoclonal antibody. This chelate must coordinate the metal under mild conditions required for the handling of antibodies, as well as provide high kinetic, thermodynamic, and metabolic stability once the metal ion is coordinated to prevent release of the radionuclide before the target site is reached in vivo. Herein, we review the role of different radiometals that have found applications of the design of radiolabeled antibodies for imaging and radioimmunotherapy. Each radionuclide is described regarding its nuclear synthesis, coordinative preference, and radiolabeling properties with commonly used and novel chelates, as well as examples of their preclinical and clinical applications. An overview of recent trends in antibody‐based radiopharma- ceuticals is provided to spur continued development of the chemistry and application of radiometals for imaging and therapy. DOI: https://doi.org/10.1002/jlcr.3590 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-167582 Journal Article Accepted Version Originally published at: Boros, Eszter; Holland, Jason P (2018). Chemical aspects of metal ion chelation in the synthesis and application antibody-based radiotracers. Journal of Labelled Compounds and Radiopharmaceuticals, 61(9):652-671. DOI: https://doi.org/10.1002/jlcr.3590 Chemical aspects of metal ion chelation in the synthesis and application antibody-based radiotracers Eszter Boros1* and Jason P. Holland2* 1 Stony Brook University, Department of Chemistry, 100 Nicolls road, 11790 Stony Brook, NY, United States 2 University of Zurich, Department of Chemistry, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland * Correspondence: Eszter Boros Tel: +1.631.632.8572 E-mail: [email protected] Jason P. Holland Tel: +41.44.63.53990 E-mail: [email protected] Running Title: Metal coordination chemistry for radiolabelling antibodies Word count (Main text): Page 1 Abstract Radiometals are becoming increasingly accessible and are utilized frequently in the design of radiotracers for imaging and therapy. Nuclear properties ranging from the emission of g-ray and b+-particles (imaging) to Auger electron, b- and a-particles (therapy) in combination with long half-lives are ideally matched with the relatively long biological half-life of monoclonal antibodies in vivo. Radiometal labelling of antibodies requires the incorporation of a metal chelate onto the monoclonal antibody. This chelate must coordinate the metal under mild conditions required for the handling of antibodies, as well as provide high kinetic, thermodynamic and metabolic stability once the metal ion is coordinated in order to prevent in vivo release of the radionuclide before the target site is reached. Herein, we review the role of different radiometals that have found applications the design of radiolabelled antibodies for imaging and radioimmunotherapy. Each radionuclide is described with regard to its nuclear synthesis, coordinative preference, and radiolabelling properties with commonly used chelates. A summary of recent developments in the area of chelate synthesis, as well as examples of their preclinical and clinical applications is presented. The aims of review are to provide an overview of recent trends in antibody-based radiopharmaceuticals and to spur continued development of the chemistry and application of radiometals for imaging and therapy. Key words Radiometals, chelates, coordination chemistry, immuno-PET, radioimmunotherapy Page 2 Introduction In the pharmaceutical industry, biological products (biologics) and related biosimilar compounds represent one of the fastest growth sectors. In 2016, the size of the biologics market was estimated to be ~$200 billion USD. Recent forecasts predict continued growth at around 10% per annum with the market reaching ~$480 billion USD by 2024. Biologics include recombinant hormones and proteins, as well as cellular- and gene-based therapies, but the majority of this growth is driven by new vaccines and monoclonal antibodies (mAbs). Monoclonal antibodies or antibody-drug conjugates (ADCs) represent the single most important branch of biologics.1 Between 2011 and August 2017, the United States Federal Drug Administration (US-FDA) approved a total of 235 New Molecular Entities (NMEs), of which 37 were either mAbs or ADCs for therapy (Figure 1). This growing trend is supported by historical data which suggest that biologics, and mAbs in particular, have received higher approval rates than conventional small-molecule drugs.2 With prescription costs for mAb-based treatments estimated to be around 22-times more than small- molecule therapies, and profit margins often over 40%, it is easy to see the commercial attraction of biologics. However, this success is partially offset by the longer time frames required from initial clinical trials to full approval, more stringent manufacturing regulations, and the associated increase in costs. Recent changes to US legislation, implemented as part of the Patient Protection and Affordable Care Act of 2010, are also helping to create an abbreviated licensure pathway for NMEs that are demonstrated to be “biosimilar” to, or “interchangeable” with, an FDA-licensed biological product. This new legal framework will speed-up the discovery and clinical translation of mAb-based agents. In nuclear medicine, desirable biochemical properties including high target affinity, specificity and selectivity, pre-optimised pharmacokinetics, and relative ease of selection using Page 3 various library technologies mean that antibody-based radiotracers are attractive platforms for developing diagnostic imaging and radioimmunotherapy (RIT) agents.3–6 Nevertheless, production and validation of radiolabelled mAbs for use as immuno-single photon emission computed tomography (immuno-SPECT), immuno-positron emission tomography (immuno-PET) or RIT agents is non-trivial.3 Synthesis of radiolabelled mAbs requires chemical reactions on the protein (Figure 2). Functionalisation usually involves post-translational modification of amino-acid side chains (particularly peptide bond formation using the primary amine group of lysine residues), derivatisation of cysteine sulfhydryl groups, or site-specific labelling of glycans using chemical and/or enzymatic methods.7,8 More recent protein engineering routes have also exploited site- specific enzymatic ligation with prominent methods including transglutaminase derivatisation, sortase coupling and formylglycine reactions to produce a range of ADCs.7–9 While the chemical nature of the linker group plays an important role in determining the metabolic stability and pharmacokinetics of a radiolabelled mAb, an equally important decision revolves around the choice of the radiometal ion and the chelation chemistry used to produce thermodynamically and kinetically stable radiometal ion complexes.10 In this review, we discuss the chemical requirements for chelation of various radiometal ions and highlight selected applications of radiometal chemistry in the development of radiotracers for immuno-PET, immune-SPECT and RIT.11 1. Radionuclides for antibody imaging and therapy In this issue of the Journal of Labelled Compounds and Radiopharmaceuticals, Engle and co- workers present an overview of the emerging radionuclides for use in combination with mAbs for diagnostic and RIT applications. Pertinent physical decay characteristics of 11 radiometal ions, and for comparison three different iodine radionuclides are presented in Table 1.12 When selecting Page 4 a radionuclide for labelling antibodies or antibody-related constructs, the most crucial decision is to ensure that the physical half-life of the radionuclide is a reasonable match to the expected 10 68 biological half-life of the radiotracer in vivo. For example, Ga (t1/2 = 67.7 min.) is an excellent radionuclide for producing PET radiotracers based on small-molecules and peptides but would be an inappropriate choice for developing immuno-PET radiotracers based on full-sized mAbs (molecular weight ~150 kDa). Experience has found that mAbs reach optimum target (usually tumour) uptake and present maximal tumour-to-background contrast ratios in mouse models at around 24 – 96 h post-administration. Hence, in this pharmacokinetic situation, 68Ga is likely to undergo complete decay before the biodistribution of a radiolabelled mAb reaches the optimum imaging window. Notably, in humans the pharmacokinetic profiles of radiolabelled mAbs are often slower than in rodents, with optimum target accumulation and image contrast achieved at time points beyond 72 h post-administration of the radiotracer. Such prolonged circulation times necessitate the use of radionuclides that have physical half-lives ranging from ca. 10 hours