Chelator compositions for radiometals and methods of using same

Abstract

A versatile chelator having the general structure (I) for chelating radiometals including lead, bismuth, actinides and lanthanides at room temperature. The chelator can be coupled to a biological targeting moiety to facilitate targeted delivery of the chelated radiometal in a mammalian subject.

Description

CHELATOR COMPOSITIONS FOR RADIOMETALS AND METHODS OF USING SAMECross-Reference to Related Applications

[0001] This application claims priority to, and the benefit of, United States provisional patent application No. 63 / 568692 filed 22 March 2024. The foregoing application is incorporated by reference in its entirety herein.Technical Field

[0002] Some embodiments relate to improved chelators. Some embodiments relate to improved biological targeting constructs incorporating chelators. Some embodiments relate to chelators coupled to a targeting moiety and capable of binding a radioactive isotope to provide targeted in vivo delivery of the radioactive isotope to a desired location within a mammalian subject.Background

[0003] As described in McNeil et al. 2021 , the fields of molecular imaging (Ml) and targeted radionuclide therapy (TRT) rely on incorporating radioisotopes onto biomolecules that show high affinity for cancer cells in order to impart diagnostic and / or therapeutic information to health researchers and clinicians. Advances in understanding the molecular processes that define normal and aberrant cell behavior has led to the identification of an increasing number of biomolecular targets that can be exploited for targeted delivery of imaging and therapeutic agents specific to diseased cells. With targeted compound delivery, one can minimize ambiguous diagnostic outcomes and / or undesirable side effects during treatment by avoiding uptake or damage induced by off-target radiopharmaceutical accumulation (Kumar Bharti et al. 2012; Bono et al. 2003). Molecular imaging relies on radionuclides which emit photons, either directly (such as in electron capture [EC] decay) or indirectly (such as in positron [|3+ ] decay), while nuclides that emit cytocidal particles (such as beta [P‘], alpha [a] particles, or Meitner-Auger electrons) can be used for TRT.

[0004] Theranostic radiopharmaceuticals represent a combination of both Ml and TRT isotopes onto a common biomolecule that can be used to both image and then treat disease, leading to a potent compound pairing that allows for visualization of the molecular processes underpinning disease and verifies cellular target presence for subsequent therapy (Yordanova et al. 2017; Rdsch et al. 2017).

[0005] In general, a theranostic pair of radionuclides comprise of two chemically similar isotopes, one which can be used for imaging, and the other for therapy (Elgqvist et al. 2014). When the theranostic pair is composed of radionuclides of two different elements, the biodistribution of the radiopharmaceutical may differ and thus any quantitative dosimetric information predicted from the diagnostic imaging results may not be reflective of the therapeutic agent; this discrepancy can be minimized with matched theranostic pairs (Elgqvist et al. 2014). Matched theranostic pairs utilize different isotopes of the same element for diagnosis and therapy, giving rise to identical chemical species and thus biodistribution, which can give further insight on the suitability of the radiopharmaceutical for a patient being assessed or treated (Yordanova et al. 2017). Only the different half lives and their effect on biodistribution may need to be considered.

[0006] Lead-203 (203Pb, ti / 2= 51.9 h) and lead-212 (212Pb, ti / 2= 10.6 h) are an elementequivalent matched theranostic pair that have generated significant interest for use in theranostic radiopharmaceutical development (Mathe et al. 2016).212Pb emits two P' particles and one a particle during its decay chain and can be used for therapy.203Pb decays by electron capture to ground state thallium-203 (203TI), followed by the emission of a gamma-photon (279 keV; 81%) that is compatible for single photon emission computed tomography (SPECT) imaging while the lack of radioactive daughter products simplifies dosimetry calculations (Horlock et al. 1975). Bismuth-212 is a daughter radionuclide of212Pb that can release one a particle and one P' particle during its decay chain through either212Po or208TI to208Pb. Having a chelator in a biological targeting vector that binds to both a desired radiometal and its daughter radionuclide may be advantageous in some circumstances, since the daughter radionuclide may be retained or rebound by the chelator.

[0007] Chelators useful in such constructs may have characteristics such as rapid complexation kinetics and strong affinity for the radionuclide under mild conditions (e.g. low temperature such as room temperature, with complexation to a high degree occurring within the span of several minutes), as well as high versatility of linker incorporation (i.e.bifunctionalization) without sacrificing the coordination integrity. While small peptidomimetics and other such constructs provide targeting moieties that may have higher tolerance for harsher radiolabeling conditions (e.g. at higher temperature), other targeting moieties such as biologies, e.g. antibodies and antigen-binding fragments thereof, may not be tolerant of harsh radiolabeling conditions such as increased temperature (e.g. may not accommodate high labelling temperatures in the range of 60°C to 90°C or higher).

[0008] Additionally, it may be desirable for chelators used in such constructs to be able to bind a plurality of different metals. Typically, a given chelator will bind well to certain metals, but not well to other metals. For example, DOTA having the following structure (A) is known as a chelator particularly for lanthanide ions. DOTAM (also referred to as TCMC) is a known chelator that has been incorporated into biological targeting constructs for carrying out targeted radiotherapy using212Pb (Stenberg et al., 2021) and has the following structure (B). However, DOTAM does not work well as a chelator for larger metals such as actinium.(A) (B)

[0009] The structure and characterization of 2,2',2",2"'-(1 ,10-dioxa-4,7,13,16- tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid, referred to as crown, is described in WO 2021 / 168567 and has the following structure (C). Crown is an effective chelator for actinium.

[0010] The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.Summary

[0011] In one aspect, a chelator having the structure (I), (II) or (III) is provided:wherein Xi and X2 are independently O, N or S and R2, R3, R4, Rs and Re are independently not present or a functional group that can be used to couple the chelator to the biological targeting moiety.

[0012] In one aspect, an in vivo radioisotope targeting construct incorporating such a chelator is provided. In one aspect, an in vivo radioisotope targeting construct containing 2 ,2',2",2"'-(1 , 10-dioxa-4,7, 13,16-tetraazacyclooctadecane-4 ,7, 13,16-tetrayl)tetraamide as a chelator is provided. In some aspects, the in vivo radioisotope targeting construct incorporates the chelator bound to a targeting moiety, optionally with a linker interposing the chelator and the targeting moiety.

[0013] In one aspect, the chelator or the in vivo radioisotope targeting construct binds to all of lead and actinium, all of lead and a lanthanide, all of lead and lutetium, all of lead and terbium, all of lead and lanthanum, all of lead, actinium and a lanthanide, all of lead, actinium, lutetium, terbium, and lanthanum, or all of lead, actinium and any one or more of lutetium, terbium or lanthanum. In one aspect, the chelator or the in vivo radioisotope targeting construct further binds bismuth. In one aspect, the binding is such that the chelator binds to each radiometal individually with a radiochemical yield of at least 20%, atleast 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature. In one aspect, a metal chelate containing such a chelator or in vivo radioisotope targeting construct bound to one or more of the foregoing metals is provided. In one aspect, the metal chelate has a charge of +3 at physiological pH when the metal is actinium or a lanthanide and the chelator is unmodified.

[0014] In one aspect, a pharmaceutical composition containing an in vivo radioisotope targeting construct as described herein in combination with a pharmaceutically acceptable carrier, excipient or vehicle is provided.

[0015] In one aspect, a method of delivering a radioisotope to a selected location within the body of a mammalian subject is provided. The method includes administering an in vivo radioisotope targeting construct as described herein to the mammalian subject, and can include allowing the targeting moiety of the in vivo radioisotope targeting construct to enhance accumulation of the radioisotope at the selected location within the body relative to other locations in the body to selectively deliver radiation to the selected location. The in vivo radioisotope targeting construct can be used to cause cell death at the selected location within the body by exposing the cells to radiation from the radioisotope, and the cells can be cancer cells. Additionally or alternatively, an imaging procedure such as PET imaging or SPECT imaging can be carried out to evaluate localization of the in vivo radioisotope targeting construct within the body, for example for a diagnostic purpose.

[0016] In one aspect, a method of delivering a radioisotope to a selected location within the body of a mammalian subject as described herein can be conducted using an in vivo radioisotope targeting construct bearing first radioisotope, and then a method of delivering a radioisotope to a selected location within the body of the mammalian subject as described herein can be conducted using the same in vivo radioisotope targeting construct bearing a second radioisotope. One of the first and second radioisotopes can be lead, and the other of the first and second radioisotopes can be an actinide such as actinium or a lanthanide such as lutetium, terbium or lanthanum. In some aspects, the other of the first and second radioisotopes can be bismuth.

[0017] In one aspect, a method of making a metal chelate using a chelator or an in vivo radioisotope targeting construct as described herein is provided. At a first time, the chelatoror the in vivo radioisotope targeting construct is combined with a first radiometal, and at a second time the chelator or the in vivo radioisotope targeting construct is combined with a second radiometal. The combining can be conducted at room temperature for both the first and second radiometals. The first and second radiometals can be lead, an actinide or a lanthanide, and the first radiometal is different from the second radiometal. One of the first and second radiometals can be lead and the other of the first and second radiometals can be actinium or a lanthanide. The lanthanide can be lutetium, terbium or lanthanum in some aspects. In some aspects, the other of the first and second radiometals can be bismuth. In some aspects, the chelator labels with both the first and second radiometals individually with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

[0018] In one aspect, a pair of metal chelates is provided using a chelator or an in vivo radioisotope targeting construct as described herein. The chelator or the in vivo radioisotope targeting construct can be bound to a first metal to provide a first metal chelate, and can be bound to a second metal to provide a second metal chelate. The binding can be conducted at room temperature. The first metal can be lead, and the second metal can be an actinide or a lanthanide. In some aspects, the second metal can be bismuth. The first metal chelate can be provided in a first solution and the second metal chelate can be provided in a second solution that is separate from the first solution. The lanthanide can be lutetium, terbium or lanthanum. The second metal chelate can have a charge of +3 at physiological pH when the second metal is an actinide or a lanthanide and the chelator is unmodified. The chelator can bind individually to each of the first and second metals with a radiochemical yield of at least 20%, at least 30%, at least 40% or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

[0019] In one aspect, a kit is provided that contains a pharmaceutical composition as described herein and instructions for combining the pharmaceutical composition with at least two radiometals. One of the at least two radiometals can be lead, and the second of the at least two radiometals can be an actinide such as actinium or a lanthanide such as lutetium, terbium or lanthanum. In one aspect, the second of the at least two radiometals can be bismuth. In one aspect, the second of the at least two radiometals is actinium andinstructions are provided for combining the pharmaceutical composition with a third radiometal that is a lanthanide such as lutetium, terbium or lanthanum. The instructions can direct that each of the combining steps be conducted at room temperature. The chelator can bind individually to each one of the at least two radiometals with a radiochemical yield of at least 20%, at least 30%, at least 40% or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

[0020] Further aspects will become apparent with reference to the following detailed description and figures illustrating exemplary embodiments.Brief Description of the Drawings

[0021] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0022] FIG. 1 shows schematically an example embodiment of an in vivo radioisotope targeting construct incorporating a chelator with a linker optionally interposing the chelator and the targeting moiety.

[0023] FIG. 2 shows the 1 H NMR spectrum and FIG. 3 shows the 13C NMR spectrum of crown amide.

[0024] FIGs. 4A and 4B show concentration dependent labelling of crown amide and DOTAM with [203Pb]Pb2+(105 kBq) in 0.1 M NH4OAc pH 7.0 buffer.

[0025] FIG. 5 shows serum stability of crown amide and DOTAM labelled with [203Pb]Pb2+.

[0026] FIG. 6 shows concentration dependent labelling of crown, crown amide and DOTAM with [225AC]AC3+(17.5 kBq) in 0.1 M NH4OAc pH 7.0 buffer.

[0027] FIG. 7 shows concentration dependent labelling of crown and crown amide with [203Pb]Pb2+(150 kBq) in 0.1 M NH4OAc pH 7.0 buffer.

[0028] FIG. 8 shows concentration dependent labelling of crown amide with [213Bi]Bi3+(20 kBq) in 0.15 M MES pH 5.5 buffer.

[0029] FIG. 9 shows concentration dependent labelling of crown amide with [155Tb]TbCl3 (50 kBq) in 0.1 M NH4OAc pH 7 buffer.

[0030] FIG. 10 shows concentration dependent labelling of crown amide with [135La]LaCl3 (100 kBq) in 0.1 M NH4OAc pH 7 buffer.

[0031] FIG. 11 shows concentration dependent labelling of crown amide with [177Lu]LuCl3 (100 kBq) in 0.1 M NH4OAc pH 7 buffer.

[0032] FIG. 12 shows the determined crystal structure of crown amide with lead.

[0033] FIG. 13 shows the determined crystal structure of crown amide with lutetium.

[0034] FIG. 14 shows concentration dependent labelling of crown amide-TATE biological targeting construct with [203Pb]PbCl2 (400 kBq) in 0.1 M NH4OAc pH 7 buffer.

[0035] FIG. 15 shows results of a human serum stability challenge of203Pb-labelled crown amide-TATE biological targeting construct.

[0036] FIG. 16 shows biodistribution of203Pb-labelled crown amide TATE and in vivo 2 hours post-injection.

[0037] FIG. 17 shows biodistribution of225Ac-labelled crown amide TATE in vivo 2 hours post-injection.Description

[0038] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0039] As used herein, the term prophylaxis includes preventing, minimizing the severity of, or preventing a worsening of a condition. As used herein, the terms treat or treatment include reversing or lessening the severity of a condition.

[0040] As used herein, the term antibody includes all forms of antibodies including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, multimeric antibodies, and the like. The term antigen bindingfragment of an antibody refers to any portion of an antibody that is capable of binding to an antigen and includes by way of example only and without limitation Fab fragments, F(ab’)2 fragments, Fv fragments, scFv fragments, minibodies, diabodies, nanobodies, affibodies, and the like. Reference to a specific antibody includes reference to any antibodies that are determined to be biosimilar to that specific antibody by any regulatory authority.

[0041] As used herein, the term peptidomimetic means a small protein-like molecule designed to mimic a peptide, and includes without limitation modified peptides, peptidic foldamers, structural mimetics and mechanistic mimetics.

[0042] A chelator composition for radiometals is disclosed. A method of using and making the composition is also disclosed. The composition can be used as a therapeutic and / or diagnostic agent.

[0043] The inventors have now determined that chelators having the general structure (1) are versatile and can coordinate radioisotopes including lead, bismuth, actinides and lanthanides under mild conditions and produce a complex that is stable under in vivo conditions, making such chelators particularly suitable for example for application in radiotherapeutic, diagnostic and / or theranostic constructs. The chelator can be coupled directly or via a linker to a biological targeting moiety to create a construct suitable for use in such applications.

[0044] The structure (1) represents 2,2', 2", 2"'-(1 , 10-dioxa-4, 7, 13, 16- tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraamide, which is referred to herein as “crown amide”. The inventors have demonstrated that crown amide is a novel effective chelator for a diverse number of metals such as lead, bismuth, actinium, and lanthanides which can coordinate under mild conditions including at room temperature and produce a stable complex in vivo.

[0045] In some embodiments, crown amide can be directly coupled to a biological targeting moiety, optionally with a linker interposing the crown amide and the biological targeting moiety, by coupling the biological targeting moiety or linker directly to one of the carboxyl groups of structure (1) to yield the structure shown as (2) below, wherein R1 is a biological targeting moiety, optionally with a linker interposing the biological targeting moiety and the crown amide chelator, illustrated as L in structure (3) below.(2) (3)In some embodiments, the crown amide chelator has the structure shown below as (4), wherein Xi and X2 are independently O, N or S. Thus, in various embodiments, the functional group provided on the bifunctional crown amide chelator to couple the chelator to the biological targeting moiety can be a carboxyl, an ester, an amide, an imide, a thioamide, a thioester, a guanidinium, or the like to yield the structure shown below as (5), wherein R1 is a biological targeting moiety, optionally with a linker interposing the biological targeting moiety and the crown amide chelator, illustrated as L in structure (6) below.

[0046] In some embodiments, the crown amide chelator is provided as a bifunctional chelator, i.e. a chelator bearing an additional functional group that can be used to couple the chelator to a targeting moiety rather than using one of the free carboxyl groups. Any suitable functional group can be coupled to structure (1) at any suitable position to yield a bifunctional chelator. For example, in some embodiments, the bifunctional chelator has the following structure (7), wherein a functional group that can be used to couple the bifunctional chelator to a biological targeting moiety to yield the structure below can be provided at one of the positions indicated by R2, R3, R4, Rs or Re, wherein R1 when present in structure (8) or (9) is a biological targeting moiety, optionally with a linker interposing the chelator and the biological targeting moiety, illustrated as L in structure (9) below.Examples of functional groups that can be used for R2, R3, R4, Rs or Re include a carboxyl, an ester, an amide, an imide, a thioamide, a thioester, a guanidinium, an ether, a thioether, an amine, or the like.(7) (8) (9)

[0047] In some embodiments, the crown amide chelator has one of the following structures (10), (11) or (12), wherein each R is independently H or a protecting group or an activating group, including for example independently the R groups listed below including Boc, Cbz or(10) (11) (12)

[0048] In one specific example embodiment, a compound having the following structure (13) is provided that enables the bifunctional chelator to be readily coupled to a biological targeting moiety while leaving all of the pendant amide groups available to coordinate with a metal:

[0049] In some embodiments as shown in FIG. 1 , an in vivo targeting chelate construct 120 has a targeting moiety 122 coupled to a chelator 126. In some embodiments, including the illustrated embodiment, a linker 124 interposes targeting moiety 122 and chelator 126.Together, targeting moiety 122, linker 124 (if present) and chelator 126 comprise in vivo targeting construct 130. Further, chelator 126 is used to chelate a radionuclide 128 that is suitable for in vivo imaging and / or radiotherapy. Radionuclide 128 together with in vivotargeting construct 130 provides an in vivo targeting chelate construct 120 suitable for targeted in vivo delivery of the radionuclide 128 payload as assisted by targeting moiety 122.

[0050] In some embodiments, a construct such as construct 120 is prepared by carrying out suitable reactions to couple targeting moiety 122 and chelator 126, for example via suitable chemical reaction, to yield an in vivo targeting construct 130, optionally with linker 124 interposing targeting moiety 122 and chelator 126. The radionuclide 128 is then added and bound to chelator 126, e.g. at a later time and in a hospital or clinic setting, to form the desired in vivo targeting metal chelate construct 120. In other embodiments, radionuclide 128 could be first chelated with chelator 126, and then chelator 126 is conjugated with targeting moiety 122 in any suitable manner to yield in vivo targeting chelate construct 120.

[0051] Any moiety suitable for directing the targeted delivery of in vivo targeting chelate construct 120 in vivo can be used as targeting moiety 122. In some embodiments, the targeting moiety 122 of the targeting construct 120 is a hapten, antigen, aptamer, affibody molecule, enzyme, protein, peptide, antibody, antigen-binding fragment of an antibody, peptidomimetic, receptor ligand, steroid, hormone, growth factor, cytokine, molecule that recognizes cell surface receptors (including molecules involved in growth, metabolism or function of cells), lipid, lipophilic group, carbohydrate, or any other molecule or targeting component capable of selectively directing a construct to a specific location within the body. The targeting moiety can be produced in any suitable manner, e.g. as a biologic, semisynthetically, or synthetically.

[0052] Examples of targeting moieties that have been developed to deliver radioisotope targeting constructs to desired locations within the body of a mammalian subject in vivo include antibodies targeting specific markers associated with specific types of cancers, peptidomimetics targeting proteins that are highly expressed in cancer cells, and the like. Exemplary non-limiting examples of suitable targeting moieties are listed in Table 1 (Lau et al. 2020). Some targeting moieties selectively interact with biological targets, including antigens, proteins, carbohydrates or other molecules present on the surface of cells that are overexpressed in cancer cells relative to normal cells, e.g. tumor-associated antigens. Exemplary non-limiting examples of suitable targets are listed in Table 1. Suitable targets and / or targeting moieties for radiopharmaceuticals, whether now known or discovered or developed in the future, would be known to a person skilled in the art. In someembodiments, targeting moiety 122 is an antibody or an antigen-binding fragment of an antibody. In some embodiments, targeting moiety 122 is a peptidomimetic. In some embodiments, the targeting moiety 122 is one of the targeting moieties listed in Table 1 , with any chelator present in the referenced molecule replaced by a crown amide chelator. In some embodiments, the targeting moiety 122 interacts selectively with one of the targets listed in Table 1.Table 1. Exemplary targeting moieties and biological targets for targeted radiation therapy.

[0053] In some embodiments, targeting moiety 122 is A33 antibody, dihydrotestosterone (DHT), HuMab-5B1 , girentuximab, AMG211 bispecific T-cell engager, IAB22M2C minibody, rituximab, obinutuzumab, U36 antibody, plerixafor, pentixafor, NFB, ipilimumab, erlotinib, PD153035, afatinib, cetuximab, panitumumab, ABY-025 affibody, HER2-nanobody, trastuzumab, pertuzumab, GSK2849330, lumretuzumab, 4FMFES, FAPI-04, FAPI-21 , FAPI-46, galactose, CB-TE2A-AR06 peptide (with crown amide substituted for DOTA), BAY 864367 peptide (with crown amide-bound ligand label instead of 18F labeling), RM2 peptide (with crown amide substituted for DOTA), SB3 peptide (with crown amide substituted for DOTA), RM26 peptide, BBN-RGD peptide, Aca-BBN peptide, NeoBOMBI peptide (with crown amide substituted for DOTA), exendin-4 peptide, glucose, codrituzumab, EF5, MISO, AZA, HX4, ASTM, LLP2A, peptidomimetic, galacto-RGD peptide, FPP(RGD)2 peptide, RGD-K5 peptide, fluciclatide, alfatide-l, alfatide-ll, PRGD2 peptide, av|36-BP peptide, CycMSHhex targeting peptides, MMOT0530A antibody, SP peptide, neurotensin, PARPi, a PSMA peptidomimetic, DCFPyL, DCFBC, HuJ591 antibody, durvalumab, nivolumab, pembrolizumab, BMS-986192 adnectin, atezolizumab, MSTP2109A antibody, TATE peptide (octreotate), TOC peptide, NOC peptide, JR11 , thymidine, fresolimumab, or bevacizumab.

[0054] In some embodiments, the biological target targeted by the in vivo radioisotope targeting construct is a tumor associated antigen, A33 transmembrane glycoprotein, androgen receptor (AR), CA19.9, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen, CD8, CD20, CD44v6, C-X-C chemokine receptor type 4 (CXCR4), cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), epidermal growth factor receptor (EGFR), epidermal growthfactor receptor 2 (ERBB2), epidermal growth factor receptor 3 (ERBB3), estrogen receptor (ER), fibroblast activation protein a, gastrin-releasing peptide receptor (GRPR), glucagon- like peptide 1 receptor (GLP-1 R), glypican 3, integrin ct4p1 , integrin av|33, integrin av|36, melanocortin-1 receptor (MC1 R), mesothelin, neurokininl receptor (NK1 R), neurotensin 1 receptor (NTS1 R), poly(ADP-ribose) polymerase 1 (PARP1), prostate-specific membrane antigen (PSMA), programmed cell death protein (PD-1), programmed death-ligand 1 (PD- L1), six-transmembrane epithelial antigen of prostate-1 (STEAP1), somatostatin receptor 2 (SSTR2), thymidine kinase, transforming growth factor-beta (TGF-P), or vascular endothelial growth factor receptor (VEGFR).

[0055] Any suitable linker can be used as linker 124 to couple chelator 126 to targeting moiety 122. For example and by way of illustration only, suitable linkers can include:• a hydrocarbon linker containing between 1 and 10 carbon atoms (C1-C10), including 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms that is optionally saturated or unsaturated, optionally substituted with one or more heteroatoms or having one or more substituents; the hydrocarbon linker can be linear, cyclic and / or branched, e.g. 8- aminooctanoic acid, 6-aminohexanoic acid;• an aromatic linker containing an aromatic moiety such as a benzyl group, e.g. aminophenylacetic acid;• an amino acid linker having between 1 and 10 amino acid residues, including 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues, any one or more of which may be naturally occurring amino acid residues, D-amino acid residues or other non-naturally occurring residues, examples of which include GlyGly, GluGluGlu, GlySerGlySer;• a cyclized linker, or cyclized ring structure, optionally a cyclized amino acid linker, e.g. aminocyclohexanecarboxylic acid;• a PEG-linker of any suitable length;• cationic linkers, whether formed from amino acid residues or other residues, e.g. Pip, 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp);• anionic linkers, whether formed from amino acid residues or other residues, e.g. AspAsp, GluGlu;• a carbohydrate containing linker;• click chemistry linkers (triazoles);• a squaramide-containing linker;• any other suitable linker;• or combinations or modifications of the foregoing.Examples of linkers that have been developed in the art for other radiopharmaceutical targeting constructs are known to those skilled in the art. Hydrophilic or charged linkers such as PEG-linkers or cationic / anionic linkers may be used to increase the overall water solubility of the targeting construct. Amino acid side chain substitutions and / or inclusion of carbohydrate moieties may be made to improve or alter the solubility and / or pharmacokinetics of the targeting construct. A person skilled in the art could develop and optimize a suitable linker for a particular application if desired. Examples of linkers that have been developed in the art for other radiopharmaceutical targeting constructs are described, by way of example only and without limitation, by Benesova et al. 2015, Benesova et al. 2016, Barnaski et al. 2017, and Kuo et al. 2018. A person skilled in the art could develop and optimize a suitable linker for a particular application.

[0056] In some embodiments, a construct such as construct 120 is prepared by carrying out suitable reactions to couple targeting moiety 122 and chelator 126, for example via suitable chemical reaction, to yield an in vivo targeting construct 130, optionally with linker 124 interposing targeting moiety 122 and chelator 126. The radionuclide 128 is then added and bound to chelator 126, e.g. at a later time and in a hospital or clinic setting, to form the desired in vivo targeting metal chelate construct 120. In other embodiments, radionuclide 128 could be first chelated with chelator 126, and then chelator 126 is conjugated with targeting moiety 122 in any suitable manner to yield in vivo targeting chelate construct 120.

[0057] In some embodiments, the radionuclide 128 is bound to chelator 126 (including as part of construct 130) under mild temperature conditions, e.g. less than about 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C or 30°C. In some embodiments, the mild temperature conditions are between about 10°C and 65°C, including any value or subrange therebetween, e.g. 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C. In some embodiments, the radionuclide 128 is conjugated to chelator 126 or construct 130 at roomtemperature, i.e. in the range of about 15°C to about 25°C, including any temperature value therebetween, e.g. 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, or 24°C.

[0058] In some embodiments, the chelator 126 or construct 130 incorporating the chelator 126 is combined with radionuclide 128 to form a metal chelate under mild pH conditions, e.g. between about 5.0 and about 7.4, including any value or subrange therebetween, e.g. 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 or 7.2. In some embodiments the radionuclide 128 is conjugated to chelator 126 at approximately neutral pH, i.e. a pH of approximately 7.0, e.g. between about 6.8 and 7.2 including any value therebetween, e.g. 6.9, 7.0 or 7.1 . In some embodiments, the radionuclide 128 is conjugated to chelator 126 at approximately physiological pH, i.e. at approximately pH 7.4, e.g. between about 7.2 and 7.6 including any value therebetween, e.g. 7.3, 7.4 or 7.5. In some embodiments, radionuclide 128 is combined with chelator 126 or construct 130 in aqueous solution. In some embodiments, the aqueous solution is free or substantially free of alcohol such as ethanol.

[0059] In some embodiments, the radionuclide 128 is combined with chelator 126 or construct 130 for an incubation period to allow a chelated metal complex to form. In some embodiments, the incubation period is between about 5 minutes and about 6 hours, including any period therebetween, e.g. 10, 15, 20, 25, 30, 45, 60 or 90 minutes, or 2, 3, 4 or 5 hours. In some embodiments, the incubation period is between about 5 minutes and about 30 minutes.

[0060] In some embodiments, the concentration of chelator 126 or construct 130 that is present when conjugated to radionuclide 128 is between about 10'4to 10'7M, including any value therebetween, e.g. 10'5or 10'6M. The concentration of chelator 126 or construct 130 that is used can be adjusted depending on the complexation kinetics between the particular chelator 126 and radionuclide 128 used in any particular embodiment. Similarly the temperature at which the radionuclide 128 is combined with chelator 126 or construct 130 can be varied depending on the complexation kinetics; generally speaking higher temperatures will increase labelling of the chelator 126 and correspondingly biological targeting construct 130 with radionuclide 128, but are undesirable where the biological targeting moiety 122 is sensitive to heat (e.g. antibodies or other proteins).

[0061] In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian serum, optionally in human serum. In some embodiments, in vivoradioisotope targeting chelate construct 120 is stable in mammalian serum, optionally in human serum. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian serum within the body of a mammal, optionally in human serum within the body of the human. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian blood, optionally in human blood. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian blood within the body of a mammal, optionally in human blood in the body of the human. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present within the body of a mammal, optionally the body of a human. In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in a mammalian cell, optionally a human cell.

[0062] In some embodiments, radionuclide 128 is delivered to a selected location within the body of a mammalian subject by administering to the subject an in vivo radioisotope targeting chelate construct 120 incorporating the radionuclide 128 and a targeting moiety 122 that specifically directs the in vivo radioisotope targeting chelate construct 120, including the bound radionuclide 128, to the selected location within the body of the subject. In some embodiments, the method includes allowing the targeting moiety 122 to enhance the accumulation of the in vivo radioisotope targeting chelate construct 120 at the selected location within the body relative to other locations in the body to selectively deliver a dose of radiation to the selected location. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is used to cause cell death at the selected location by delivering a targeted dose of radiation from the radionuclide 128. In some embodiments, the cells that are killed at the selected location are cancer cells. In some embodiments, the radiation is alpha radiation and / or beta radiation. Meitner-Auger electrons or gamma radiation could be used in other embodiments.

[0063] In some embodiments, the cancer cells that are killed at the selected location are colorectal cancer, prostate cancer, pancreatic cancer, bladder cancer, clear cell renal cell carcinoma, gastrointestinal adenocarcinoma, melanoma, lung cancer, hepatocarcinoma, 13- cell lymphoma, head and neck cancer, hematological malignancies, solid malignancies, melanoma, nonsmall cell lung carcinoma, breast cancer, gynecologic cancer, ovarian cancer, glioma, insulinoma, neoplasm, pancreatic ductal adenocarcinoma, neuroendocrine tumors, or the like.

[0064] In some embodiments, in vivo radioisotope targeting chelate construct 120 is internalized by a cell within the mammalian subject, for example by endocytosis or otherwise. Thus in some embodiments, in vivo radioisotope targeting chelate construct 120 is present within a mammalian cell. In some embodiments, the in vivo radioisotope targeting chelate construct 120 is present within a human cell.

[0065] In some embodiments, the in vivo radioisotope targeting chelate construct 120 is prepared prior to administration of construct 120 to a subject by combining an in vivo radioisotope targeting construct 130 having a targeting moiety 122, a chelator 126 and optionally a linker 124 with a radionuclide 128 to form the in vivo radioisotope targeting chelate construct 120. In some embodiments, the combining is carried out at a mild temperature, e.g. at a temperature in the range of about 10°C to about 65°C, including any value therebetween e.g. 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C. In some embodiments, the combining is carried out at room temperature, e.g. at a temperature in the range of about 15°C to about 25°C, including any value therebetween e.g. 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, or 24°C. In some embodiments, the combining is carried out at a mild pH, e.g. an approximately neutral pH or an approximately physiological pH. In some embodiments, the mild pH is a pH of between about 5.0 and about 7.4, including any value therebetween e.g. 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 or 7.4. In some embodiments, the mild pH is approximately 6.0. In some embodiments, the combining is carried out a physiological pH, e.g. in the range of about. 7.0 to 7.4 including any value therebetween, e.g. 7.1 , 7.2 or 7.3. In some embodiments, radionuclide 128 is combined with in vivo radioisotope targeting construct 130 in aqueous solution. In some embodiments, the aqueous solution is free or substantially free of alcohols such as ethanol. In some embodiments, the combining is carried out for a period of between about 5 and about 30 minutes, including any value therebetween e.g. 10, 15, 20 or 25 minutes.

[0066] In some embodiments, in vivo targeting chelate construct 120 is used in diagnostic applications. For example, in vivo targeting chelate construct 120 may be administered to a subject in any suitable manner, and any suitable imaging technology or procedure may be used to evaluate the localization of the targeting chelate construct 120 within the body via targeting moiety 122 by visualizing the location of bound radionuclide 128, e.g. positron emission tomography (PET) imaging or single-photon emission computerized tomography(SPECT) imaging. Such imaging procedures can be carried out for example to diagnose a subject as having a particular disorder or type of cancer, or to localize regions of the subject’s body affected by the particular disorder or type of cancer. In some embodiments, localization of targeting chelate construct 120 to a target organ, region or plurality of loci within the body as evaluated by such imaging technology may be indicative that the subject has a particular form of cancer, and / or can be used to evaluate the extent of the cancer and or locations within the body wherein cancerous cells are or may be located, and / or can be used to evaluate the extent of metastasis of the cancer or the decrease in cancer cells as treatment proceeds or the like.

[0067] In some embodiments, constructs such as targeting chelate construct 120 are used in therapeutic applications, for example to carry out targeted radionuclide therapy. For example, targeting chelate construct 120 may be administered to a subject in any suitable manner, and the targeting effect imparted by targeting moiety 122 can be used to deliver the chelated radionuclide 128 to a desired location within the subject’s body. In some embodiments, radiation from radionuclide 128 is used to kill cells at the desired location. In some embodiments, the cells that are killed at the desired location are cancer cells. In some embodiments, targeting construct 120 is used to perform targeted radionuclide therapy. In some embodiments, targeting construct 120 is used to perform targeted radiotherapy, including targeted alpha therapy and / or targeted beta therapy.

[0068] In some embodiments, in vivo targeting construct 130 can be combined with two different radiometals to provide two different targeting constructs 120 that can be used at different times or for different purposes. For example, in vivo targeting construct 130 may be combined with a first radiometal at a first time and the resultant metal chelate targeting construct 120 used to carry out a diagnostic or imaging procedure, or to carry out a first round of targeted radiotherapy for therapeutic purposes. In vivo targeting construct 130 may subsequently be combined with a second radiometal at a second time, and the resultant metal chelate targeting construct 120 can be used to carry out a round of targeted radiotherapy for therapeutic purposes, either subsequent to an initial diagnostic procedure or subsequent to a first round of targeted radiotherapy. In some embodiments, the first and second radiometals are different metals, and at least one of the first and second radiometals is lead while the other of the first and second radiometals is an actinide such as actinium or a lanthanide such as lutetium, terbium, lanthanum, or the like, or bismuth. Insome embodiments, in vivo targeting construct 130 can be labelled with both the first and second radiometals at room temperature.

[0069] In some embodiments, a pharmaceutical composition is provided, the pharmaceutical composition comprising a construct such as targeting construct 120 and a pharmaceutically acceptable carrier. The pharmaceutical composition may include any suitable excipient, vehicle, buffer, diluent, binder, thickener, lubricant, preservative or the like, and may be provided in any desired state, e.g. as a liquid, suspension, emulsion, paste, or the like. In some embodiments, the pharmaceutical composition can be administered in any suitable manner, e.g. orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intratumorally, by inhalation, or the like.

[0070] In some embodiments, a kit containing such a pharmaceutical composition and instructions for combining the pharmaceutical composition with a radiometal is provided. In some embodiments, the instructions are instructions for combining the pharmaceutical composition with at least two different radiometals. In some embodiments, the instructions are for forming two different solutions, each one of the two different solutions containing a different one of the at least two different radiometals. In some embodiments, the instructions direct that each one of the two different solutions be formed at different times. In some embodiments, the instructions are for combining the pharmaceutical composition with at least two different radiometals selected from lead, bismuth, an actinide and a lanthanide. In some embodiments, one of the radiometals is lead and a second one of the radiometals is actinium or a lanthanide such as lutetium, terbium or lanthanum. In some embodiments, one of the radiometals is lead, a second one of the radiometals is actinium, and the kit further includes instructions for combining the pharmaceutical composition with a lanthanide such as lutetium, terbium or lanthanum. In some embodiments, one of the radiometals is lead, and a second one of the radiometals is bismuth. In some embodiments, the instructions specify that the pharmaceutical composition should be combined with each of the at least two different radiometals at room temperature.

[0071] In some embodiments, a method of prophylaxis and / or treatment of a subject having or believed to have cancer is provided. In some embodiments, the method comprises administering an in vivo targeting chelate construct 120 or a pharmaceutical composition comprising such a targeting chelate construct 120 to the subject. In some embodiments,the method comprises administering a therapeutically and / or prophylactically effective amount of the targeting chelate construct 120 to the subject.

[0072] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In alternative embodiments, the subject is livestock or a pet, e.g. a horse, cow, sheep, goat, cat, dog, rabbit, or the like. In some embodiments, the subject is a monkey.

[0073] While exemplary embodiments are described herein with reference to the targeting and killing of cancer cells, such constructs can be used for the selective killing and / or ablation of other undesired cell types, for example bacteria, fungi, cells implicated in autoimmune disorders, virus-infected cells, parasites, and so on.

[0074] In some embodiments, the metals that can be used as radionuclide 128 include lead or actinium or a lanthanide or bismuth. In some embodiments, the metal is a radioisotope. In some embodiments, the radioisotope is any desired radioisotope, for example an actinide, a lanthanide, lead, bismuth, or the like. In some embodiments, the actinide is actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium. In some embodiments, the lanthanide is lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, or lutetium. In some embodiments, the radioisotope is212Pb,203Pb,225AC, or the like. In some embodiments, the radioisotope is212Pb / 203Pb,225Ac,155Tb / 161Tb,135La / 132La / 133La,177Lu,212Bi / 213Bi or the like.

[0075] In some embodiments, a versatile chelator or radioisotope targeting construct incorporating such a versatile chelator that can chelate a plurality of different metals to provide a metal chelate at room temperature is provided. In some embodiments, the plurality of different metals include lead and one other metal selected from an actinide and a lanthanide or bismuth. In some embodiments, the plurality of different metals include an actinide and lead. In some embodiments, the plurality of different metals include an actinide, a lanthanide, and lead. In some embodiments, the plurality of different metals include actinium and lead. In some embodiments, the plurality of different metals include actinium, lead, and one or more lanthanides. In some embodiments, the plurality of different metals include lead and one or more lanthanides. In some embodiments, the plurality of different metals include actinium, lutetium and lead. In some embodiments, theplurality of different metals include actinium, terbium and lead. In some embodiments, the plurality of different metals include actinium, lanthanum and lead. In some embodiments, the plurality of different metals include lutetium and lead. In some embodiments, the plurality of different metals include terbium and lead. In some embodiments, the plurality of different metals include lanthanum and lead. In some embodiments, the plurality of different metals include actinium, lead, lutetium, terbium and lanthanum. In some embodiments, the plurality of different metals further include bismuth. In some embodiments, the plurality of different metals include lead and bismuth.

[0076] Without being bound by theory, in some embodiments providing a versatile chelator that can be used to chelate a plurality of different metals at room temperature can streamline the manufacture of a radioisotope targeting construct incorporating the chelator and / or can allow for greater flexibility in the use of the radioisotope targeting construct in a healthcare setting. For example, sometimes it is desirable to administer two different radioisotopes to a patient, whether using one as a diagnostic agent to visualize the distribution of a target within the patient’s body (e.g. the location and / or relative number of cancer cells) and a second as a therapeutic agent to kill the target cells within the patient’s body, or using two different therapeutic radionuclides that emit different types of radiation to kill target cells within the patient’s body (e.g. tandem therapy with lutetium and actinium as is used to treat late stage prostate cancer using PSMA as a targeting agent (Khreish et al., 2020)). Or one radioisotope may be more readily available in a healthcare setting at any given time than another radioisotope, meaning flexibility in choosing which radioisotope to use may facilitate patient treatment. In such cases, having a single radioisotope targeting construct containing a single chelator that can bind to both radiometals, rather than requiring two different radioisotope targeting constructs each containing a different chelator that can bind to each of the radiometals individually but not to both, can streamline manufacture, distribution and use of the radioisotope targeting construct.

[0077] In some embodiments, a chelator that binds to a radiometal is considered to be a chelator that labels with a radiochemical yield of at least 20% with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate (NF OAc) buffer at pH 7.0. In some embodiments, a chelator that binds to a radiometal at room temperature is considered to be a chelator that labels with a radiochemical yield of at least 20% with at least 10 kBq of the radiometal when the chelatoris present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature. In some embodiments, a chelator that binds to a radiometal is considered to be a chelator that labels with a radiochemical yield of at least 30% under the foregoing conditions. In some embodiments, a chelator that binds to a radiometal is considered to be a chelator that labels with a radiochemical yield of at least 40% under the foregoing conditions. In some embodiments, a chelator that binds to a radiometal is considered to be a chelator that labels with a radiochemical yield of at least 50% under the foregoing conditions.

[0078] In some embodiments, the chelator or radioisotope targeting construct incorporating the chelator binds to all of lead, an actinide and a lanthanide to form a metal chelate. In some embodiments, the chelator or radioisotope targeting construct incorporating the chelator further binds to bismuth to form a metal chelate. In some embodiments, at a first time a first metal chelate is formed by combining the chelator or radioisotope targeting construct incorporating the chelator with a first radiometal, and at a second time that is later than the first time, a second metal chelate is formed by combining the chelator or radioisotope targeting construct incorporating the chelator with a second radiometal. In some embodiments, said combining is conducted at room temperature. In some embodiments, a pair of metal chelates is provided using the same chelator or radioisotope targeting construct incorporating the chelator, the first metal chelate is provided by combining the chelator or radioisotope targeting construct incorporating the chelator with a first metal and the second metal chelate is provided by combining the chelator or radioisotope targeting construct incorporating the chelator with a second metal, wherein the first and second metals are lead, bismuth, an actinide or a lanthanide, wherein the first metal is not the same as the second metal and wherein at least one of the first and second metals is lead. In some embodiments, the first and second metal chelates are provided in separate solutions. In some embodiments, said combining is conducted at room temperature.

[0079] In some embodiments, the chelator is bound to a metal ion to form a coordination complex. In some embodiments, the coordination complex is referred to as a metal chelate. In some embodiments, the metal chelate or crown amide as the chelating ligand is associated with one or more cations as counter ions, for example Na+, K+, Ca2+or the like. In some embodiments, the metal chelate or the chelating ligand is fully protonated. In someembodiments, the metal chelate or the chelating ligand is neutrally charged. In some embodiments, the metal chelate or the chelating ligand is in a partially protonated state.

[0080] In some embodiments, the unmodified chelator or unmodified in vivo radioisotope targeting construct has a neutral charge and is bound to a metal ion to form a coordination complex that has a positive charge at physiological pH. For example, in one embodiment, the unmodified chelator or targeting construct is bound to Ac3+and the resulting construct has a charge of +3 at physiological pH. In one embodiment, the unmodified chelator or targeting construct is bound to a lanthanide such as La3+, Tb3+, or Lu3+and the resulting construct has a charge of +3 at physiological pH. In one embodiment, the unmodified chelator is bound to lead Pb2+and the resulting construct has a charge of +2 at physiological pH. Without being bound by theory, it is believed that a metal chelate that has a positive charge may have a different biological distribution of the metal chelate than would be the case for a metal chelate that has a neutral or negative charge (see e.g. Renard et al., 2021). In some embodiments, the unmodified chelator or in vivo radioisotope targeting construct is bound to the metal ion to form the coordination complex at room temperature. While in some cases chemical modifications can be made to a chelator or to an in vivo radioisotope targeting construct incorporating the chelator to provide a desired charge, the crown amide chelator described herein being neutrally charged at physiological pH can be used to provide a metal chelate that is positively charged at physiological pH without a need to carry out such modifications to the structure of the chelator or the in vivo targeting construct. This is in contrast to other chelators having carboxylate pendant arms such as DOTA, which will tend in their unmodified state to deprotonate and be negatively charged at physiological pH, thereby neutralizing the positive charge of the radiometal or even potentially introducing a charge of -1 at physiological pH depending on the functionalization of the chelator or the in vivo radioisotope targeting construct.

[0081] In some embodiments, the coordination complex is present in mammalian serum, optionally human serum. In some embodiments, the coordination complex is stable in mammalian serum, optionally human serum. In some embodiments, the coordination complex is present in mammalian serum within the body of the mammal, optionally present in human serum within the body of the human. In some embodiments, the coordination complex is present in blood, optionally human blood. In some embodiments, the coordination complex is stable in mammalian blood, optionally human blood. In someembodiments, the coordination complex is present in mammalian blood within the body of the mammal, optionally present in human blood within the body of the human. In some embodiments, the coordination complex is present within the body of a mammal, optionally present within the body of a human. In some embodiments, the coordination complex is present within a cell of a mammalian subject, optionally present within a cell of a human subject.

[0082] Without being bound by theory, the examples described herein demonstrate that crown amide as a chelator is highly versatile for binding a range of radiometals, particularly larger radiometals, including lead, actinium, bismuth, the exemplary lanthanides terbium, lanthanum and lutetium, including e.g.212Pb / 203Pb,225Ac,155Tb / 161Tb,135La / 132La / 133La,177Lu and213Bi / 212Bi. Crown amide is particularly versatile because it can be labelled at room temperature with all of the foregoing range of radiometals.

[0083] Furthermore, the inventors found that crown amide effectively chelated the desired radiometals with good stability over several days at 37°C in human serum. This suggests that crown amide is likely to effectively chelate the desired radiometals in in vivo applications, e.g. when administered to a mammalian subject for imaging and / or therapeutic purposes.

[0084] Thus, from the examples described herein, it can be soundly predicted that crown amide can be used as a chelator for the in vivo delivery of radioisotopes including lead, bismuth, actinides and lanthanides for the conduct of targeted radiotherapy or imaging when conjugated to a targeting moiety that targets the vector to a suitable location in vivo.Examples

[0085] Specific embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in scope.Example 1 .0 - Synthesis and Characterization of Crown Amide

[0086] Synthesis of N,N'-(ethane-1,2-diyl)bis(4-methylbenzenesulfonamide), S1 : 300 mmol (20 mL, 18 g, 1 eq.) of ethylene diamine was added to 1 L (0.3M) pyridine cooled to - 20°C. Next, 617 mmol (117 g, 2.05 eq) of 4-methylbenzylsulfonyl chloride was added slowly over ten minutes with vigorous stirring. After the addition was complete the reaction was allowed to warm to room temperature and react overnight (15 hrs). 850 mL of the pyridine solvent was then removed via distillation, and the remaining reaction mixture was poured into 300 mL of room temperature water with fast stirring. The product S1 precipitated as an off-white powder which was further dried under heating and vacuum to yield 108 g of an off- white solid.1H NMR (300 MHz, DMSO-d6): 5 7.61 - 7.57 (m, 4H), 7.36 (d, 4H), 2.73 - 2.65 (m, 4H), 2.37 (s, 6H). ESI MS: [M+Py+H]+: 448.1(S1) (S2)

[0087] Synthesis of 4,7,13,16-tetratosyl-1,10-dioxa-4,7,13,16-tetraazacyclooctadecane, S2: 212 mmol (78.2 g, 1 eq.) of S1 was added to 150 mL of dry DMF along with 355 mmol (49 g, 1 .67 eq.) of K2CO3, and 210 mmol (30 g, 24.6 mL, 1 eq) of Bis-chloroethyl ether. The reaction was set to reflux with a drying tube and allowed to react overnight (15 hrs). Upon completion the solvent was removed via rotary evaporation to yield a dark brown gum. To the reaction flask was added 200 mL of acetone, the flask was then set for reflux and the gum slowly dispersed in the acetone. The reaction mixture was then filtered while hot and the filter cake was transferred back into the boiling flask. This process was repeated once more with fresh acetone and then the acetone washings were discarded. The solid was washed with 200 mL of boiling deionized water and the mixture was filtered while hot. This was repeated one more time before the water washings were discarded. The remaining filter cake was an off white solid. The product was then transferred to a new flask and dried under heat and vacuum to yield 16 g (17%) of an off white amorphous solid.1H NMR (400 MHz, CDCI3, 298K): 5 7.73 (8H, d), 7.34 (8H, d), 3.56 (8H, t), 3.35(8H, s), 3.24 (8H, t), 2.45 (12H, s). ESI MS: [M+H]+: 877.6, [M+Na]+ 899.6(S2) (S3)

[0088] Synthesis of 1,10-dioxa-4,7,13,16-tetraazacyclooctadecane, S3: 33 mmol (29 g, 1 eq.) of S2 and 300 mmol (28 g, 9 eq.) of phenol was added to a 1 L round bottom flask. The flask was equipped with a reflux condenser and was purged with Ar. Next 250 mL of 33% w / w HBr in acetic acid was added to the flask at 0°C via syringe. The reaction was then allowed to reflux for 48 hours before it was cooled then poured into 1 L of diethyl ether. The HBr salt precipitated as a black powder, which was then washed with acetone and dried to yield 18.8 grams (98%) of and off white solid, S3 4HBr. The HBr salt was then free based by dissolving in a minimal amount of aqueous NaOH ensuring the final pH of the solution was > 12. The aqueous solution was then extracted 5 times with CHCh. The organic layers were combined and dried over Na2SO4, and the solvent was removed to yield 6.0 g (70% overall) of S3 as an off white waxy solid.1H NMR (400 MHz, D2O, 298K) 5 3.87 (8H, t), 3.58 (8H, s), 3.40 (8H, t). ESI MS: [M+H]+: 261.3(S3) crown amide (1)

[0089] Synthesis of 2,2',2",2'"-(1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetamide, Crown-Amide (1): 3.4 mmol (870 mg, 1 eq.) of S3 and16.6 mmol (2.3 g 4.9 eq.) of 2-Bromoacetamide, were added to a round bottom flask along with 18.7 mmol (2.6 ml, 5.5 eq.) of triethylamine and 26 mL of ethanol. The reaction was allowed to reflux for 4 hours where a white precipitate formed. The precipitate was filtered and washed with cold ethanol to yield 1 .03 g (62%) of crown-amide as a white powder.1HNMR (600 MHz, D2O, 298K) 6: 4.33 (s, 8H), 3.98 (t, J= 4.3 Hz, 8H), 3.87 (s, 8H), 3.69 (m, 8H) (FIG. 2).13C NMR (150 MHz, D2O, 298K) 5: 168.4, 63.6, 57.2, 53.5, 50.4 (FIG. 3). HRMS ESI+: [M+Na]+511.2962.1.) Oxalyl Chloride

[0090] Synthesis of tert-butyl (2-bromoacetyl)carbamate: Synthesis of tert-butyl (2- bromoacetyl)carbamate was conducted according to the literature process described by Leonard et al. 1985. 7.24 mmol (1 g, 1 eq.) of 2-bromoacetamide was added to a round bottom flask along with 3.5 mL of DCE. The flask was cooled to 0°C and purged with argon before the addition of 8.70 mmol (0.74 mL, 1.2 eq.) of oxalyl chloride. The reaction was allowed to stir at 0°C for 10 minutes before being heated to 65°C for 2.5 hours. The reaction mixture was then re-cooled to 0°C before the addition of 14.5 mmol (1 .38 mL, 2 eq.) of tBuOH in 3 ml of DCE (1 ,2-dichloroethane). The reaction mixture was allowed to stir for 25 minutes cold before being poured into an ice-cold solution of saturated NaHCOa. The organic layer was then separated and washed with more saturated NaHCOa, followed by deionized water. The organic layer was then dried over Na2SO4 and the solvent was removed under vacuum, to yield 1.25 g (72%) of an off white powder.1H NMR (400 MHz, CDCI3, 298K) 5 7.53 (br. s, 1 H), 4.30 ( s, 2H), 1 .51 (s, 9H).

[0091] Synthesis of 2-(7,13,16-tris(2-((tert-butoxycarbonyl)amino)-2-oxoethyl)-1,10- dioxa-4,7,13,16-tetraazacyclooctadecan-4-yl)acetic acid: 1 mmol (1 g, 1 eq.) of 2- chlorotrityl chloride resin was swelled with DCM (dichloromethane) and mixed with 5 mmol (0.700 g, 5 eq) of bromoacetic acid in 10 mL of DCM and 1 .25 mL DIPEA (N,N-diisopropylethylamine, 7 mmol, 7 eq). The mixture was allowed to shake for 1 hr before the the solvent was removed and the resin was washed 3 times with 10 mL of DCM. The resin was then capped by stirring with 10 mL of a 17:2:1 DCM:MeOH:DIPEA mixture for 0.5 hrs. Next the remaining capping solution was drained and the resin was rinsed 3X with 10 mL of DCM. Next 4 mmol (1 ,04g, 4 eq.) of S3 was added to the resin in 5 mL of CHCI3 and 1 .75 mL DIPEA (9.8 mmol, 9.8 eq.). The resin was then shaken at 40°C for 18 hrs before the solvent was removed and the resin was rinsed 3 times with 10 mL of DCM. Next 5 mmol (1 .2 g, 5 eq.) was added to the resin in 5 mL of CHCI3 and 2 mL DIPEA (11 .2 mmol, 11 .2 eq.). The resin was allowed to shake for 24 hrs at 40°C. Next the excess solvent was removed, and the resin was washed 3 times with 10 mL of DCM. The resin was then cleaved by shaking the resin with 5 mL of 2:3 Trifluoroethanol: DCM for 2 hrs the mixture was filtered and the solution phase was collected. The solvent was then removed to afford a yellow powder. Which was further purified by HPLC to afford the product. Example 1.1 - Proposed Synthesis of Bifunctional Crown Amide Structure (13)

[0092] A synthetic scheme was devised to synthesize compound (13), a bifunctional derivative of crown amide which without being bound by theory will allow crown amide to be coupled to a biological targeting moiety while leaving all of the pendant amino acid arms free to chelate the metal.Example 2.0 - Concentration Dependent Radiolabelling of Pb

[0093] 10 pL of 10'3M aqueous crown amide, 10 pL of 1.0 M NH4OAC pH 7 buffer, 70 pL of H2O and 10 pL of [203Pb]PbCl2 (105 kBq), was mixed in 0.6 mL Eppendorf tubes and allowed to react for 60 mins. After 60 mins at room temperature, 3 pL of the reaction was spotted onto SA iTLC plates which were subsequently developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated Pb2+remained at baseline (Rf<0.2) and free Pb2+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (no ligand being used), and compared to a known standard DOTAM for a positive control. The process was repeated such that experiments with final [crown amide] were 10’4M, 10’5M, 10’6M, 10’7M, and 10’8M.

[0094] Results are shown in FIGs. 4A and 4B, with FIG. 4A showing the radiochemical yield versus the concentration of each ligand and FIG. 4B showing the radiochemical yield versus the ratio of ligand:metal.Example 3.0 - LogD74 Determination for Pb

[0095] 30 pL (4.23 MBq) of [203Pb]PbCI2, 5 pL of 10’3M crown amide and 15 pL of 1 .0 M NH4OAC pH 7 buffer was mixed and allowed to react at room temperature for 30 mins. Equivalent conditions were used for DOTAM. Quantitative labelling was verified via SA iTLC (see above). With quantitative labelling achieved a 15 pL aliquot of the reaction mixture was mixed with 485 pL of pH 7.4 phosphate buffered saline. Next 500 pL of n-octanol was added and the mixture was vortexed for 5 mins. The aqueous layer and octanol layer were then separated via centrifuge, and activity in each layer was quantified via gamma spectroscopy. Results are shown in Table 2 and show that crown amide has good hydrophilicity.Table 2. LogD?.4 values for [203Pb]Pb-Crown Amide and [203Pb]Pb-DOTAM.Example 4.0 - Human Serum Stability Study for Pb

[0096] 5 pL of 10'3M aqueous crown amide, 5 pL of 1.0 M NH4OAC pH 7 buffer, 1 pL of [203Pb]PbCl2 (338 kBq / pL), and 39 pL of deionized H2O was mixed and allowed to react at room temperature for 30 minutes. Quantitative labelling was achieved and assessed via iTLC. The reaction was then diluted with 50 pL of human serum, and set to incubate on a shaking plate at 37°C. The radiolabelling was assessed at ~ 24hr, 48hr, and 72 hr timepoints via iTLC. A negative control with 5 pL of deionized H2O added instead of ligand was also evaluated. Results are shown in FIG. 5 and confirm that crown amide exhibits good serum stability when labelled with [203Pb]Pb2+over several days.Example 5.0 - Comparative Study of Binding Properties of Crown versus Crown Amide for Ac and Pb

[0097] 10 pL of 10'3M aqueous crown or crown amide, 10 pL of 1 .0 M NH4OAC pH 7.0 buffer, 10 pL of [225Ac]AcCl3 (17.5 kBq), and 70 pL of deionized water were mixed in 1.5 mL Eppendorf tubes and allowed to react at room temperature for 60 mins. After 60 mins, 5 pL of the reaction was spotted onto SA iTLC plates which were subsequently developed in 50 mM pH 5 EDTA. The plates were then scanned with a Radio TLC Scanner, under such conditions chelated Ac3+remained at baseline (Rf<0.2) and free Ac3+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (10 pL of deionized water added instead of crown amide or crown). The process was repeated such that experiments with final concentrations 10'4M, 10'5M, 10'6M, 10'7M for crown amide and crown, and 10'4M, 10'5M for DOTAM. Under those conditions, crown amide showed good binding affinity (quantitative labeling at 10'6M chelator concentration), similar to crown, while DOTAM does not effectively label225Ac3+(FIG. 6).

[0098] Without being bound by theory, the observed crown amide labeling with225Ac is surprising because it is contradictory to the belief that Ac3+as a ‘hard’ metal ion has requires ‘hard’ donors such as carboxylic acids. In fact, crown amide seems to be the only non-carboxylic acid containing chelator for actinium reported so far (Thiele et al., 2018).

[0099] 10 pL of 10'4M aqueous crown or crown amide, 10 pL of 1 .0 M NH4OAC pH 7.0 buffer, 2 pL of [203Pb]PbCl2 (150 kBq), and 78 pL of deionized water were mixed in 1.5 mL Eppendorf tubes and allowed to react at room temperature for 60 mins. After 60 mins, 5 pL of the reaction was spotted onto SA iTLC plates which were subsequently developed in 50 mM pH 5 EDTA. The plates were then scanned with a Radio TLC Scanner, under suchconditions chelated Pb2+remained at baseline (Rf<0.2) and free Pb2+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (10 pL of deionized water added instead of the chelator). The process was repeated such that experiments with final concentrations, 10'5M, 10'6M, 10'7M for crown amide, and 10'4M, 10'5M, 10'6M, 10'7M for crown. Under those conditions, crown amide showed high binding affinity (>90% labeling at 10'6M chelator concentration), while crown does not bind with Pb2+(FIG. 7). In a separate comparison experiment as described above, DOTAM showed good binding affinity with Pb2+, although not as high as crown amide (FIGs. 4A and 4B).Example 6.0 - Concentration Dependent Radiolabelling of Bi

[0100] 3pL of 10'3M aqueous crown amide, 10 pL of 0.5 M MES pH 5.5 buffer, and 20 pL of [213Bi]BiCl3 (20 kBq), were mixed in 1.5 mL Eppendorf tubes and allowed to react for 15 mins. After 15 mins at room temperature, 5 pL of the reaction was spotted onto SA iTLC plates which were subsequentially developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated Bi3+remained at baseline (Rf<0.2) and free Bi3+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (no ligand being used with 3 pL of deionized water added instead). The process was repeated such that experiments with final concentrations 10'4M, 10'5M, 10'6M, for crown amide. Results are shown in FIG. 8 showing the radiochemical yield versus the concentration of crown amide.Example 7.0 - Concentration Dependent Radiolabelling of Tb-155

[0101] 5 pL of 10'3M aqueous crown amide, 5 pL of 1.0 M NH4OAC pH 7 buffer, and 2 pL of [155Tb]TbCl3 (50 kBq), and 38 pL of deionized H2O, was mixed in 0.6 mL Eppendorf tubes and allowed to react for 60 mins. After 60 mins at room temperature, 3 pL of the reaction was spotted onto SG iTLC plates which were subsequentially developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated Tb3+remained at baseline (Rf<0.2) and free Tb3+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (no ligand being used). The process was repeated such that experiments with final crown amide concentrations were 10'4M, 10'5M, 10'6M, and 10'7M.Results are shown in FIG. 9 and confirm that crown amide binds well to the exemplary lanthanide terbium and could be effectively labelled at room temperature.of La- 135

[0102] 5 pL of 10'3M aqueous crown amide, 5 pL of 1.0 M NH4OAC pH 7 buffer, 4 pL of [135La]LaCl3 (100 kBq), and 36 pL of deionized water was mixed in 0.6 mL Eppendorf tubes and allowed to react for 60 mins. After 60 mins at room temperature, 3 pL of the reaction was spotted onto SG iTLC plates which were subsequentially developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated La3+remained at baseline (Rf<0.2) and free La3+migrated to the solvent front (Rf~ 1). The process was verified by negative control (no ligand being used). The process was repeated such that experiments with final crown amide concentrations were 10'4M, 10'5M, 10'6M, and 10'7M. Results are shown in FIG. 10 and confirm that crown amide binds well to the exemplary lanthanide lanthanum and could be effectively labelled at room temperature.Example 9.0 - Concentration Dependent Radiolabelling of Lu-177

[0103] 5 pL of 10'3M aqueous crown amide, 5 pL of 1.0 M NH4OAC pH 7 buffer, 1 pL of [177Lu]LuCl3 (100 kBq), and 39 pL of deionized water was mixed in 0.6 mL Eppendorf tubes and allowed to react for 60 mins. After 60 mins at room temperature, 3 pL of the reaction was spotted onto SG iTLC plates which were subsequentially developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated Lu3+remained at baseline (Rf<0.2) and free Lu3+migrated to the solvent front (Rf~ 1). The process was verified by negative control (no ligand being used. The process was repeated such that final crown amide concentrations were 10'4M, 10'5M, 10'6M, and 10'7M. Results are shown in FIG. 11 and confirm that crown amide binds well to the exemplary lanthanide lutetium and could be effectively labelled at room temperature.Example 10.0. Determination of Crown Amide-Pb Crystal Structure

[0104] Molar equivalents of crown amide and Pb(NO3)2 were dissolved in 200 pL of deionized H2O before the addition of 400 pL of isopropyl alcohol was added and mixed well.Next 300 pL of diethyl ether was gentle layered on top of the solution and the mixture was allowed to stand until crystal formation occurred. A suitable crystal with dimensions 0.31 x 0.21 x 0.12 mm3was selected and mounted on a mylar loop oil on a Bruker APEX-II CCD diffractometer. The crystal was kept at a steady T = 100(2) K during data collection. The structure was solved with the SheIXT (Sheldrick, 2015) solution program using Intrinsic Phasing methods and by using Olex2 1.5 (Dolomanov et al., 2009) as the graphical interface. The model was refined with ShelXL 2019 / 3 (Sheldrick, 2015) using full matrix least squares minimisation on F2. The crystal structure is shown in FIG. 12 and confirms the mode of binding of Pb to crown amide.Example 11 .0. Determination of Crown Amide-Lu Crystal Structure

[0105] Molar equivalents of Crown Amide and Lu(NOa)3 were dissolved in 200 pL of deionized H2O before the addition of 400 pL of isopropyl alcohol was added and mixed well. Next 300 pL of diethyl ether was gently layered on top of the solution and the mixture was allowed to stand until crystal formation occurred. A suitable crystal with dimensions 0.24 x 0.18 x 0.15 mm3was selected and mounted on a mylar loop in oil on a Bruker D8 VENTURE diffractometer. The crystal was kept at a steady T = 100(2) K during data collection. The structure was solved with the XT (Sheldrick, 2015) solution program using Intrinsic Phasing methods and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with XL (Sheldrick, 2015) using full matrix least squares minimisation on F2. The crystal structure is shown in FIG. 13 and confirms the mode of binding of Lu to crown amide.Example 12.0 - Synthesis of Crown Amide-TATE Biological Targeting Construct.

[0106] Synthesis of Crown Amide-Tate. Uncyclised TATE peptide was synthesized via solid phase peptide synthesis starting from Wang resin functionalized with Fmoc-Thr(OtBu)- OH. First the Fmoc group was removed with soaking in 20% piperidine in dimethylformamide (condition a). Then the next amino acid Fmoc-Cys(Acm)-OH was coupled using 3 eq of amino acid, 3 eq of HBTU, and 3 eq of HOBT, 15 eq diisopropylethylamine in 4 mL dimethylformamide and 1 mL dichloromethane (condition b).This was process of Fmoc removal (a) followed by amino acid coupling (b) was repeated for remaining amino acids in the peptide sequence.Next crown-tris(Boc-amide) (2-(7,13,16-tris(2-((tert-butoxycarbonyl)amino)-2-oxoethyl)-1 ,10- dioxa-4,7,13,16-tetraazacyclooctadecan-4-yl)acetic acid) was coupled to the resin using 2 eq. of crown-tris(Boc-amide), 2 eq. of HBTU, 2eq of HOBT, and 15 eq. of diisopropylethylamine in 4 mL of dimethylformamide and 1 mL of dichloromethane (condition c). Next the resin was cyclized by mixing the resin with 15 eq. of h in 0.5 mL dimethylformamide and 0.2 mL H2O (condition d). Finally the peptide was cleaved from the resin and all acid labile protecting groups were removed using 3 mL of a 90:5:5 triflouroacetic acid: triisopropylsilane: H2O solution. The crown amide-TATE product was then purified via HPLC with 20:80 Acetonitrile (0.1 % TFA):H2O (0.1 % TFA) mobile phase. Synthesis of the desired construct was confirmed by ESI MS (positive mode): [M+2H]2+=760.8 , [M+3H]3+=507.6.Example 13.0 - Radiolabelling of Crown Amide-TATE with Pb

[0107] 5 pL of 10'3M aqueous crown amide-TATE, 5 pL of 1.0 M NH4OAC pH 7 buffer, and 1 pL of [203Pb]PbCl2 (400 kBq), and 39 pL of deionized H2O was mixed in 0.6 mL Eppendorf tubes and allowed to react for 60 mins. After 60 mins at room temperature, 3 pL of the reaction was spotted onto SA iTLC plates which were subsequentially developed in 50 mM pH 5 EDTA. The plates were then scanned, under such conditions chelated Pb2+remained at baseline (Rf<0.2) and free Pb2+migrated to the solvent front (Rf ~ 1). The process was verified by negative control (no ligand being used), and compared to a known standard DOTAMTATE for a positive control. The process was repeated such that final crown amide- TATE concentrations were 10'4M, 10'5M, 10'6M, and 10'7M. The results are shown in FIG. 14 and demonstrate that crown amide-TATE is capable of high radiolabelling efficiency with lead down to concentrations as low as 1 pM at room temperature.Example 14.0 - LogD74 Determination of r203Pb1Pb- Crown Amide-TATE

[0108] 8 pL (3.2 MBq) of [203Pb]PbCI2, 5 pL of 10’3M crown amide-TATE, 4 pL of 1.0 M NH4OAC pH 7 buffer and 23 pL of deionized H2O was mixed and allowed to react at room temperature for 30 mins. Quantitative labelling was verified via SA iTLC (see above). With quantitative labelling achieved a 13pL aliquot of the reaction mixture was mixed with 487 pL of pH 7.4 phosphate buffered saline. Next 500 pL of n-octanol was added to and themixture was vortexed for 5 mins. The aqueous layer and octanol layer were then separated via centrifuge, and activity in each layer was quantified via gamma spectroscopy. Results are shown in Table 3. These results demonstrate that [203Pb]Pb-crown amide-TATE is very hydrophilic.Example 15.0 - Human Serum Stability Challenge of Pb-labelled Crown Amide-TATE

[0109] 5 pL of 10'3M aqueous crown amide-TATE, 5 pL of 1.0 M NH4OAC pH 7 buffer, 1 pL of [203Pb]PbCl2 (386 kBq / pL), and 39 pL of DI H2O was mixed and allowed to react at room temperature for 30 minutes. Quantitative labelling was achieved and assessed via SA iTLC. The reaction was then diluted with 50 pL of human serum, and set to incubate on a shaking plate at 37°C. The %Bound activity was assessed at ~4hr, 24hr, and 52 hr, timepoints via iTLC. Process was repeated for DOTAMTATE. Results are shown in FIG. 15.Example 16.0 - Biodistribution of Pb-labelled Crown Amide-TATE in vivo

[0110] 8.2 pL [203Pb]PbCI2(36.2 MBq), 30 pL 10’4M crown amide-TATE, and 4 pL of 1.0 M NH4OAC pH 7 buffer were mixed and allowed to react at room temperature for 30 mins, and quantitative radiolabelling was assessed by SA iTLC. 1.3 pL of this solution (1.07 MBq, 0.1 nmol), was aliquoted, added to 9 pL 10'4M crown amide-TATE an then diluted to 996 pL with USP grade saline to make the dosing solution of [203Pb]Pb-crown amide-TATE.

[0111] To each mouse (male NOD.Cg-Rag1tm1 Mom H2rgtm1Wjl / SzJ (NRG), previously inoculated with AR42J derived cells on left shoulder) 100 pL (100 kBq, 0.1 nmol of [203Pb]Pb- Crown Amide-TATE) each of this dosing solution was administered via a tail vein injection using a Tailveiner restrainer. After 2 hours all mice were humanely euthanized (n = 4 per time point) by CO2 asphyxiation under 5% isoflurane anaesthesia, followed by cardiac puncture. Tumour and other organs were then collected from each mouse and weighed before being counted in a gamma counter to determine the quantity of [203Pb]Pb-crown amide-TATE in each organ as % injected activity / gram. Results are shown in FIG. 16.Example 17.0 - Radiolabelling of Crown Amide-TATE with Ac

[0112] Crown amide-TATE showed superior Ac-225 radiolabelling efficiency to DOTATATE where crown amide-TATE labelled 20 kBq of Ac-225 (2*10'5M ligand concentration) quantitatively in 30 mins at room temperature but DOTATATE could only achieve 25 % radiochemical yield for a 60 min reaction at 80 °C, with the activity and ligand concentration being the same. Additionally, crown amide-TATE can easily achieve an apparent molar activity of 300 kBq / nmol where as DOTATATE struggles to achieve >100 kBq / nmol reliably.Example 18.0 - Biodistribution of Ac-labelled Crown Amide-TATE in vivo

[0113] 20 pL 10’4M crown amide-TATE, 61 .2 pL [225Ac]AcCI3(600 kBq) and 8 pL 1 M NH4ACO buffer were added to a 1.5 mL eppendorf tube. The reaction was allowed to react for 60 minutes before quantitative radiolabelling was confirmed with iTLC. The reaction mixture was then loaded onto 10 mg of C-18 resin pre-equilibrated with 2 mL of ethanol followed by 3 ml of aqueous saline. The resin was washed with 3 mL of saline then the [225Ac]Ac-Crown Amide-TATE was eluted with 90:10 EthanokSaline (v / v) solution. A portion of this elute was diluted to 1330 pL with saline to serve as the dosing solution of the [225Ac]Ac-Crown Amide-TATE.

[0114] To each mouse (male NOD.Cg-Rag1tm1 Mom H2rgtm1Wjl / SzJ (NRG), previously inoculated with AR42J derived cells on left shoulder) 100 pL (29.3 kBq, 0.1 nmol of [225Ac]Ac-Crown Amide-TATE) each of this dosing solution was administered via a tail vein injection using a Tailveiner restrainer. After 2 hours all mice were humanely euthanized (n = 4 per time point) by CO2 asphyxiation under 5% isoflurane anaesthesia, followed by cardiac puncture. Tumour and other organs were then collected from each mouse and weighed and allowed to sit for 24 hours before being counted in a gamma counter to determine the quantity of [225Ac]Ac-crown amide-TATE in each organ as % injected activity / gram. Results are shown in FIG. 17. For comparison purposes, results of the equivalent experiment with [225Ac]Ac-crown-TATE are shown in Ingham et al., 2024.

[0115] The foregoing examples establish that crown amide is a good chelator for lead, actinium, bismuth and the exemplary lanthanides terbium, lutetium and lanthanum, and would therefore have good utility as a chelator for use in an in vivo targeting vector delivering these radiometals or other actinides or lanthanides.

[0116] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.References

[0117] The following references are of interest with respect to the subject matter herein, and are hereby incorporated by reference herein in their entireties:• Baranski, A.-C.; Schafer, M.; Bauder-Wust, U.; Wacker, A.; Schmidt, J.; Liolios, C.; Mier, W.; Haberkorn, U.; Eisenhut, M.; Kopka, K.; Eder, M., Improving the Imaging Contrast of68Ga-PSMA-11 by Targeted Linker Design: Charged Spacer Moieties Enhance the Pharmacokinetic Properties. Bioconjugate Chem. 2017, 28, 2485-2492.• Benesova, M.; Schafer, M.; Bauder-Wust, U.; Afshar-Oromieh, A.; Kratochwil, C.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M., Preclinical Evaluation of a Tailor- Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer. J. Nucl. Med. 2015, 56, 914-920.• Benesova, M.; Bauder-Wust, U.; Schafer, M.; Klika, K. D.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M., Linker Modification Strategies To Control the Prostate- Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors. J. Med. Chem. 2016, 59, 1761-1775.• Bono J, Tolcher A, Rowinsky E. The future of cytotoxic therapy: selective cytotoxicity based on biology is the key. Breast Cancer Res. 2003, 5 3 154-9.• Dolomanov O.V. and L.J. Bourhis and R.J. Gildea and J.A.K. Howard and H. Puschmann, Olex2: A complete structure solution, refinement and analysis program, J. Appt. Cryst., (2009), 42, 339-341.• Elgqvist J, Frost S, Pouget J-P, Albertsson P. The potential and hurdles of targeted alpha therapy - clinical trials and beyond. Front Oncol. 2014, 3:324.• Horlock P, Thakur M, Watson I. Cyclotron produced lead-203. Postgrad Med J. 1975, 57:751-4.• Ingham et al., Nucl. Med. Biol. 2024:138-139:108944.• Khreish, F., Ebert, N., Ries, M. et al.225Ac-PSMA-617 / 177Lu-PSMA-617 tandem therapy of metastatic castration-resistant prostate cancer: pilot experience. EurJ Nucl Med Mol Imaging 47, 721-728 (2020).• Kumar Bharti S, Banerjee T, Brosh RM. Setting the stage for cohesion establishment by the replication fork. Cell Cycle. 2012; 77:2114-35.• Kuo, H.-T.; Pan, J.; Zhang, Z.; Lau, J.; Merkens, H.; Zhang, C.; Colpo, N.; Lin, K.-S.; Benard, F. Effects of linker modification on tumor-to- kidney contrast of68Ga- labeled PSMA-targeted imaging probes. Mol Pharm. 2018, 15, 3502-3511.• Lau, J.; Rousseau, E.; Kwon, D.; Lin, K.; Benard, F.; Chen, X., Insight into the Development of PET Radiopharmaceuticals for Oncology. Cancers 2020, 12, 1312.• Leonard, J.; Cruickshank, K.A. Nucleoside annelating reagents: N-(tert- butoxycarbonyl)-2-bromoacetamide and 2-chloroketene diethyl acetal. J. Org. Chem. 1985, 50, 14, 2480-2488.• McNeil, B.L., Robertson, A.K.H., Fu, W. et al. Production, purification, and radiolabeling of the203Pb / 212Pb theranostic pair. 2021 , EJNMMI radiopharm. chem. 6, 6.• Mathe D, Szigeti K, Hegedus N, Horvath I, Veres DS, Kovacs B, Szucs Z. Production and in vivo imaging of203 Pb as a surrogate isotope for in vivo212 Pb internal absorbed dose studies. Appl Radiat Isot. 2016, 114:1-6.• Renard et al., Positron Emission Tomography Imaging of Neurotensin Receptor- Positive Tumors with68Ga-Labeled Antagonists: The Chelate Makes the Difference Again, J. Med. Chem., 2021 64(12), 8564-8578• Rdsch F, Herzog H, Qaim SM. The beginning and development of the theranostic approach in nuclear medicine, as exemplified by the radionuclide pair86Y and90Y. Pharmaceuticals. 2017, 10(2): 1-28.• Sheldrick, G.M., Crystal structure refinement with ShelXL, Acta Cryst., (2015), C71, 3-8.• Sheldrick, G.M., SheIXT-lntegrated space-group and crystal-structure determination, Acta Cryst., (2015), A71 , 3-8.• Stenberg VY, Larsen RH, Ma LW, Peng Q, Juzenas P, Bruland 0S, Juzeniene A. Evaluation of the PSMA-Binding Ligand212Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer. IntJ Mol Sci. 2021 , 22(9):4815.• Thiele NA, Wilson JJ. Actinium-225 for Targeted a Therapy: Coordination Chemistry and Current Chelation Approaches. Cancer Biother Radiopharm. 2018, 33(8):336- 348.• Yordanova A, Eppard E, Kurpig S, Bundschuh RA, Schdnberger S, Gonzalez- Carmona M, Feldmann G, Ahmadzadehfar H, Essler M. Theranostics in nuclear medicine practice. OncoTargets Therapy. 2017, 70:4821-8.

Claims

CLAIMS:1 . A chelator having the structure (I), (II) or (III):(I) (ID (III) wherein:Xi and X2 are independently O, N or S;R2, R3, R4, Rs and Re are independently not present or a functional group that can be used to couple the chelator to the biological targeting moiety.

2. An in vivo radioisotope targeting construct comprising a chelator as defined in claim 1 coupled to a biological targeting moiety.

3. The in vivo radioisotope targeting construct as defined in claim 2 comprising the following structure (IV), (V) or (VI):(IV) (V) (VI) wherein at least one of R2, R3, R4, Rs and Re is present, and wherein R1 represents the biological targeting moiety.

4. The in vivo radioisotope targeting construct as defined in claim 3 comprising the following structure (VII), (VIII) or (IX):(VII) (VIII) (IX) wherein L represents a linker.

5. The in vivo radioisotope targeting construct as defined in any one of claims 2 to 4, wherein only one of R2, R3, R4, Rs and Re is present.

6. The chelator or the in vivo radioisotope targeting construct as defined in any one of claims 1 to 5, wherein R2, R3, R4, Rs or Re when present are independently a carboxyl, an ester, an amide, an imide, a thioamide, a thioester, a guanidinium, an ether, a thioether, or an amine group.

7. The in vivo radioisotope targeting construct as defined in any one of claims 3 to 6, wherein the linker L when present comprises a C1-C10 hydrocarbon linker that is optionally substituted with one or more heteroatoms or has one or more substituents, an aromatic linker, a cationic linker, an anionic linker, an amino acid linker having between one and ten amino acids, a cyclized amino acid linker, a PEG linker, a cyclized ring linker, an aromatic linker, a squaramide linker or a click chemistry linker.

8. The chelator or an in vivo radioisotope targeting construct as defined in any one of claims 1 to 7, wherein the chelator binds to the following metals: all of lead and actinium; all of lead and a lanthanide; all of lead and lutetium; all of lead and terbium;all of lead and lanthanum; all of lead, an actinide, and a lanthanide; all of lead, actinium and a lanthanide; all of lead, actinium, lutetium, terbium, and lanthanum; all of lead, actinium, and lutetium; all of lead, actinium and terbium; or all of lead, actinium and lanthanum, with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least50%, with at least 10 kBq of each metal individually when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

9. The chelator or the in vivo radioisotope targeting construct as defined in any one of claims 1 to 8, further comprising a radiometal chelated by the chelator to form a metal chelate, optionally wherein: the radiometal comprises an actinide, a lanthanide, or lead; or the radiometal comprises actinium, lead, terbium, lanthanum, or lutetium.

10. The chelator or the in vivo radioisotope targeting construct as defined in claim 9, wherein the radiometal comprises actinium, terbium, lanthanum or lutetium, and wherein the metal chelate has a charge of +3 at physiological pH when the chelator or the in vivo radioisotope targeting construct is unmodified.1 1 . The in vivo radioisotope targeting construct as defined in any one of claims 2 to 10, wherein the targeting moiety comprises a hapten, an antigen, an aptamer, an affibody, an enzyme, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody, a peptidomimetic, a receptor ligand, a steroid, a hormone, a growth factor, a cytokine, a molecule that recognizes cell surface receptors, a lipid, a lipophilic group, or a carbohydrate.

12. The in vivo radioisotope targeting construct as defined in claim 11 , wherein the antigen-binding fragment of an antibody comprises an Fab fragment, an F(ab’)2 fragment, a Fv fragment, an scFv fragment, a minibody, a diabody, a nanobody, or an affibody.

13. The in vivo radioisotope targeting construct as defined in any one of claims 2 to 12, wherein the biological targeting moiety comprises A33 antibody, dihydrotestosterone (DHT), HuMab-5B1 , girentuximab, AMG211 bispecific T-cell engager, IAB22M2C minibody, rituximab, obinutuzumab, U36 antibody, plerixafor, pentixafor, NFB, ipilimumab, erlotinib, PD153035, afatinib, cetuximab, panitumumab, ABY-025 affibody, HER2-nanobody, trastuzumab, pertuzumab, GSK2849330, lumretuzumab, 4FMFES, FAPI-04, FAPI-21 , FAPI-46, galactose, CB-TE2A-AR06 peptide (with crown amide substituted for DOTA), BAY 864367 peptide (with crown amide-bound ligand label instead of 18F labeling), RM2 peptide (with crown amide substituted for DOTA), SB3 peptide (with crown amide substituted for DOTA), RM26 peptide, BBN- RGD peptide, Aca-BBN peptide, NeoBOMBI peptide (with crown amide substituted for DOTA), exendin-4 peptide, glucose, codrituzumab, EF5, MISO, AZA, HX4, ASTM, LLP2A, peptidomimetic, galacto-RGD peptide, FPP(RGD)2 peptide, RGD-K5 peptide, fluciclatide, alfatide-l, alfatide-ll, PRGD2 peptide, av|36-BP peptide, CycMSHhex targeting peptides, MMOT0530A antibody, SP peptide, neurotensin, PARPi, a PSMA peptidomimetic, DCFPyL, DCFBC, HuJ591 antibody, durvalumab, nivolumab, pembrolizumab, BMS-986192 adnectin, atezolizumab, MSTP2109A antibody, TATE peptide (octreotate), TOC peptide, NOC peptide, JR11 , thymidine, fresolimumab, or bevacizumab.

14. The in vivo radioisotope targeting construct as defined in any one of claims 2 to 13, wherein a biological target targeted by the in vivo radioisotope targeting construct comprises: a tumor associated antigen, A33 transmembrane glycoprotein, androgen receptor (AR), CA19.9, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen, CD8, CD20, CD44v6, C-X-C chemokine receptor type 4 (CXCR4), cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 (ERBB2), epidermal growth factor receptor 3 (ERBB3), estrogen receptor (ER), fibroblast activation protein a, gastrinreleasing peptide receptor (GRPR), glucagon-like peptide 1 receptor (GLP-1 R), glypican 3, integrin ct4(31 , integrin av|33, integrin av|36, melanocortin-1 receptor (MC1 R), mesothelin, neurokininl receptor (NK1 R), neurotensin 1 receptor (NTS 1 R), poly(ADP-ribose) polymerase 1 (PARP1), prostate-specific membrane antigen (PSMA), programmed cell death protein (PD-1), programmed death-ligand 1 (PD-L1), six-transmembrane epithelial antigen of prostate-1 (STEAP1), somatostatin receptor 2 (SSTR2), thymidine kinase, transforming growth factor-beta (TGF-P), or vascular endothelial growth factor receptor (VEGFR).

15. An in vivo radioisotope targeting construct comprising 2,2',2",2"'-(1 ,10-dioxa- 4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraamide as a chelator.

16. A pharmaceutical composition comprising an in vivo radioisotope targeting construct as defined in any one of claims 2 to 15 and a pharmaceutically acceptable carrier, excipient or vehicle.

17. A method of delivering a radioisotope to a selected location within the body of a mammalian subject, the method comprising: administering an in vivo radioisotope targeting construct as defined in any one of claims 2 to 15 bearing the radioisotope to the mammalian subject.

18. The method as defined in claim 17, further comprising allowing the targeting moiety of the in vivo radioisotope targeting construct to enhance accumulation of the radioisotope at the selected location within the body relative to other locations in the body to selectively deliver radiation to the selected location.

19. The method as defined in either one of claims 17 or 18, further comprising a step of forming a chelate comprising the radioisotope and the in vivo radioisotope targeting construct prior to the administering step, wherein the step of forming the chelate construct comprises combining the in vivo radioisotope targeting construct with the radioisotope at a temperature of between about 10°C and about 25°C for an incubation period, optionally wherein the temperature is between about 15°C and about 25°C during the incubation period and optionally wherein the incubation period is between about 5 minutes and about 30 minutes.

20. The method as defined in any one of claims 17 to 19, wherein the combining step is carried out at a pH in the range of about 5.0 to about 7.4.21 . The method as defined in any one of claims 17 to 20, further comprising carrying out an imaging procedure to evaluate localization of the in vivo radioisotope targetingconstruct within the body, wherein the imaging procedure optionally comprises positron emission tomography (PET) imaging or single-photon emission computerized tomography (SPECT) imaging.

22. The method as defined in any one of claims 17 to 21 , wherein the in vivo radioisotope targeting construct is used to cause cell death at the selected location within the body by exposing the cells to radiation from the radioisotope.

23. The method as defined in claim 22, wherein the in vivo radioisotope targeting construct is used to cause death of cancer cells at the selected location within the body, optionally wherein the cancer cells are colorectal cancer, prostate cancer, pancreatic cancer, bladder cancer, clear cell renal cell carcinoma, gastrointestinal adenocarcinoma, melanoma, lung cancer, hepatocarcinoma, B-cell lymphoma, head and neck cancer, hematological malignancies, solid malignancies, melanoma, nonsmall cell lung carcinoma, breast cancer, gynecologic cancer, ovarian cancer, glioma, insulinoma, neoplasm, pancreatic ductal adenocarcinoma, or neuroendocrine tumors.

24. The method as defined in either one of claims 22 or 23, wherein the radiation comprises alpha radiation, beta radiation or both alpha radiation and beta radiation.

25. The method as defined in any one of claims 17 to 24, wherein the mammalian subject is a human.

26. The method as defined in any one of claims 17 to 25 that is conducted using a first radioisotope, the method further comprising, at a later time, administering the same in vivo radioisotope targeting construct as defined in any one of claims 2 to 15 bearing a second radioisotope to the mammalian subject, wherein one of the first and second radioisotopes is lead and the other of the first and second radioisotopes is actinium or a lanthanide.

27. A method of forming a metal chelate comprising combining a chelator having the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) below with a radiometal in an aqueous solution at a temperature of between 15°C and 25°Cwherein:Xi and X2 are independently O, N or S;R2, R3, R4, Rs and Re are independently not present or a functional group that can be used to couple the chelator to a biological targeting moiety;R1 when present represents a biological targeting moiety; andL when present represents a linker.

28. The method as defined in claim 27, wherein only one of R2, R3, R4, Rs and Re is present.

29. The method as defined in any one of claims 27 to 28, wherein the aqueous solution comprises a pH in the range of about 5.0 to about 7.4 or wherein said combining step is conducted for a period of between about 5 and about 30 minutes.

30. The method as defined in any one of claims 27 to 29, wherein the radiometal comprises actinium or a lanthanide, and wherein the metal chelate has a charge of +3 at physiological pH when the chelator is unmodified.31 . The method as defined in any one of claims 27 to 30, wherein: at a first time, said combining is conducted with a first radiometal, the first radiometal comprising lead, an actinide or a lanthanide; and at a second time that is later than the first time, said combining is conducted again with a second radiometal, the second radiometal comprising lead, an actinide or a lanthanide; wherein the first radiometal is not the same as the second radiometal and wherein one of the first and second radiometals comprises lead.

32. The method as defined in claim 31 , wherein the actinide comprises actinium and wherein the lanthanide comprises lutetium, terbium or lanthanum.

33. The method as defined in either one of claims 31 or 32, wherein the chelator labels with both the first radiometal and the second radiometal individually with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

34. The method as defined in any one of claims 31 to 33, wherein: the first radiometal comprises lead and the second radiometal comprises actinium; the first radiometal comprises actinium and the second radiometal comprises lead; the first radiometal comprises lead and the second radiometal comprises lutetium; the first radiometal comprises lutetium and the second radiometal comprises lead; the first radiometal comprises lead and the second radiometal comprises terbium;the first radiometal comprises terbium and the second radiometal comprises lead; the first radiometal comprises lead and the second radiometal comprises lanthanum; or the first radiometal comprises lanthanum and the second radiometal comprises lead.

35. A pair of metal chelates comprising a chelator or an in vivo radioisotope targeting construct as defined in any one of claims 1 to 15, the pair of metal chelates comprising: the chelator or the in vivo radioisotope targeting construct bound to a first metal to provide a first metal chelate; and the chelator or the in vivo radioisotope targeting construct bound to a second metal to provide a second metal chelate; wherein the first metal comprises lead, and wherein the second metal comprises an actinide or a lanthanide; wherein the chelator binds individually to each of the first and second metals with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

36. The pair of metal chelates as defined in claim 35, wherein the first metal chelate is provided in a first solution and the second metal chelate is provided in a second solution.

37. The pair of metal chelates as defined in any one of claims 35 to 36, wherein the actinide comprises actinium and wherein the lanthanide comprises lutetium, terbium or lanthanum.

38. The pair of metal chelates as defined in any one of claims 35 to 37, wherein the second metal chelate has a charge of +3 at physiological pH when the chelator or the in vivo radioisotope targeting construct is unmodified.

39. The pair of metal chelates as defined in any one of claims 35 to 38, wherein: the first metal comprises lead and the second metal comprises actinium;the first metal comprises lead and the second metal comprises lutetium; the first metal comprises lead and the second metal comprises terbium; or the first metal comprises lead and the second metal comprises lanthanum.

40. A kit comprising a pharmaceutical composition as defined in claim 16 and instructions for combining the pharmaceutical composition with at least two radiometals to form at least two solutions, a first one of the at least two solutions comprising the in vivo targeting construct chelated to a first one of the at least two radiometals and a second one of the at least two solutions comprising the in vivo targeting construct chelated to a second one of the at least two radiometals.41 . The kit as defined in claim 40, wherein the at least two radiometals comprise: actinium and lead; lutetium and lead; terbium and lead; lanthanum and lead; actinium, lutetium and lead; actinium, terbium and lead; actinium, lanthanum and lead; actinium, terbium, lutetium and lead; actinium, terbium, lanthanum and lead; actinium, lutetium, lanthanum and lead; actinium, lutetium, terbium and lead; or actinium, lutetium, terbium, lanthanum and lead.

42. The kit as defined in any one of claims 40 or 41 , wherein when the pharmaceutical composition is combined with actinium, lutetium, terbium or lanthanum, the resultant metal chelate has a charge of +3 at physiological pH when the in vivo radioisotope targeting construct is unmodified.

43. The kit as defined in any one of claims 40 to 42, wherein the instructions direct that the first and second ones of the at least two solutions be prepared at different times.

44. The kit as defined in any one of claims 40 to 43, wherein the instructions direct that the first and second solutions be prepared at room temperature.

45. The kit as defined in any one of claims 40 to 44, wherein the chelator of the in vivo radioisotope targeting construct binds individually to each one of the at least two radiometals with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

46. The chelator or the in vivo radioisotope targeting construct as defined in any one of claims 1 to 7, wherein the chelator binds to the following metals: all of lead and bismuth; all of lead, actinium and bismuth; or all of lead, a lanthanide and bismuth; wherein the lanthanide optionally comprises lutetium, terbium or lanthanum.

47. The chelator or the in vivo radioisotope targeting construct as defined in any one of claims 1 to 8, further comprising a radiometal chelated by the chelator to form a metal chelate, wherein the radiometal comprises bismuth.

48. The method as defined in any one of claims 17 to 25 that is conducted using a first radioisotope, the method further comprising, at a later time, administering the same in vivo radioisotope targeting construct as defined in any one of claims 2 to 15 bearing a second radioisotope to the mammalian subject, wherein one of the first and second radioisotopes is lead and the other of the first and second radioisotopes is bismuth.

49. The method as defined in any one of claims 27 to 30, wherein: at a first time, said combining is conducted with a first radiometal, the first radiometal comprising lead or bismuth; and at a second time that is later than the first time, said combining is conducted again with a second radiometal, the second radiometal comprising lead or bismuth;wherein the first radiometal is not the same as the second radiometal, optionally wherein the chelator labels with both the first radiometal and the second radiometal individually with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

50. A pair of metal chelates comprising a chelator or an in vivo radioisotope targeting construct as defined in any one of claims 1 to 15, the pair of metal chelates comprising: the chelator or the in vivo radioisotope targeting construct bound to a first metal to provide a first metal chelate; and the chelator or the in vivo radioisotope targeting construct bound to a second metal to provide a second metal chelate; wherein the first metal comprises lead, and wherein the second metal comprises bismuth; optionally wherein the chelator binds individually to each of the first and second metals with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.51 . The kit as defined in claim 40, wherein the at least two radiometals comprise bismuth and lead, optionally wherein the chelator of the in vivo radioisotope targeting construct binds individually to each one of the at least two radiometals with a radiochemical yield of at least 20%, at least 30%, at least 40%, or at least 50%, with at least 10 kBq of the radiometal when the chelator is present at a concentration of 10 pM in 0.1 M ammonium acetate buffer at pH 7.0 at room temperature.

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