1,7,13-trioxa-4,10,16-triazacyclooctadecane chelator compositions for radiometals and methods of using same

Abstract

A chelator having the general structure (I) for chelating radiometals such as 225Ac or 161 / 155 / 149 / 152Tb under mild conditions is provided. The chelator can be coupled to a biological targeting moiety to facilitate targeted delivery of the chelated radiometal in a mammalian subject.

Description

1,7,13-TRIOXA-4,10,16-TRIAZACYCLOOCTADECANE 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 / 625221 filed 25 January 2024 and entitled CHELATOR COMPOSITIONS FOR RADIOMETALS AND METHODS OF USING SAME and to United States provisional patent application No. 63 / 626696 filed 30 January 2024 and entitled NOVEL OCTREOTATE-CHELATOR. Both of the foregoing applications are incorporated by reference herein in their entireties.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] In the realm of modem medicine, the integration of nuclear techniques has flourished, offering innovative solutions for diagnosing and treating various diseases. Among these, targeted radionuclide therapy stands out as a promising avenue, harnessing the power of specific isotopes to selectively eradicate malignant cells.1The success of such therapeutic strategies hinges on the development of efficient chelators, molecular entities designed to tightly bind and carry radionuclides to their intended targets.

[0004] Targeted alpha therapy (TAT) is an emerging medical approach that utilizes alphaemitting radioactive isotopes to selectively destroy cancer cells while minimizing damage to surrounding healthy tissues.2 225Ac has emerged as a leading candidate for TAT due to its favorable half-life (9.92 days) and potent therapeutic decay progeny (four alpha particles emitted during its decay, FIG. 1).2To advance its clinical translation, the development ofhigh-denticity chelating ligands is imperative for stable chelation of [225Ac]Ac3+ions, with the added capability of sequestering imaging isotopes for diagnostic assessments.

[0005] The radio-lanthanide ion [155Tb]Tb3+presented itself as a promising imaging counterpart with bonding characteristics similar to [225Ac]Ac3+(coordination number and ionic radii).3 4This makes it suitable for SPECT / CT diagnostics, given its low-energy gamma emissions and a half-life of 5.32 days. Additionally, [155Tb]Tb3+contributes to the "terbium theranostic quartet," comprising radioisotopes149Tb,152Tb,155Tb, and161Tb.4This quartet spans both PET and SPECT imaging modalities, along with all three therapeutic decay types (a, p- and Meitner-Auger electrons), presenting a compelling avenue for developing a theranostic radiopharmaceutical with equivalent pharmacokinetic and biodistribution properties in the field of nuclear medicine.

[0006] Macrocyclic chelating ligands exhibit enhanced kinetic inertness and thermodynamic stability, qualities imperative for / n vivo applications. Nevertheless, achieving these characteristics frequently comes at the cost of specificity and selectivity, and the process often demands elevated temperatures for the incorporation of metal ions, as seen with the widely used chelator DOTA.5

[0007] Historically, non-macrocyclic chelators like DTPA exhibited fast kinetics but suffered from in vivo kinetic lability. More recently, a new class of non-macrocyclic chelators with enhanced inertness has emerged, incorporating bidentate picolinic acid pendant arms with a larger backbone, thereby improving their effectiveness for larger metals such as [225Ac]Ac3+(H4py4pa, H4picoopa) and, more recently, for [155Tb]Tb3+(H4noneunpa).3’6’7

[0008] In the past few years, researchers, including Wilson et al. (macropa, macrodipa, py- and py2-macrodipa) and Yang et al. (crown), have been at the forefront of developing macrocyclic chelators with expanded backbones based on 18-membered macrocycles.5’8-13These structural features offer improved complexation kinetics, facilitating fast complexation under mild conditions, making them compatible with temperature-sensitive targeting vectors.

[0009] In a recent study, Wilson et al. investigated the complexation capabilities of two chelators, macrotripa and macrodipa, using the triaza and diaza-18-crown-6 backbone, respectively, with the non-radioactive lanthanide series. Notably, both chelatorsdemonstrated "dual size selectivity," showing an ability to preferentially bind both large and small lanthanides.9

[0010] Targeting constructs have been developed that utilize a targeting moiety that targets a desired region of the body (e.g. a tumor-associated antigen) covalently coupled to a chelator to secure radionuclides for such purposes. The targeting moiety can be coupled to the chelator via a linker. Such targeting constructs may be referred to as radioimmunoconjugates. The radioimmunoconjugate is used to chelate a desired radionuclide for in vivo delivery, for example to provide diagnostic imaging, targeted radionuclide therapy using the construct, or both (i.e. as a theranostic construct).

[0011] 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).

[0012] Ideally, Ac-radiopharmaceuticals should have low normal tissue uptake and fast tumor internalization to help mitigate any cytotoxicity induced by irradiation of healthy tissue, and ensure alpha-emitting daughter radionuclides released from the targeting vector are contained inside the tumors. An effective chelator that does not release the bound radiometal readily under physiological conditions is important to achieving this.Chart 1. Structures of currently available chelators.

[0013] The structure and synthesis of 2,2',2"-(1 ,7,13-trioxa-4, 10,16-triazacyclooctadecane-4, 10, 16-triyl)triacetic acid has been described in Chen et al., Inorg. Chem.

[0014] 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

[0015] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the abovedescribed problems have been reduced or eliminated, while other embodiments are directed to other improvements.

[0016] One aspect of the invention provides a chelator having the structure (I) or an in vivo radioisotope targeting construct comprising a biological targeting moiety and a chelator having the structure (I):

[0017] One aspect of the invention provides a chelator or an in vivo radioisotope targeting construct, having one of the following structures (121) or (124) wherein each Ri is independently H or an alkyl amine having n carbon atoms wherein n is an integer between 1 and 4, a benzyl group with a reactive functional group (e.g., amine, hydroxyl, isothiocyanate) attached to the benzyl at the para, ortho or meta position, and * indicates chirality when R1 is not H,or having the following structure (127) wherein R3 is an hydroxyl group or an amine group and R2 is C, S or N

[0018] In some aspects, the chelator or in vivo radioisotope targeting construct comprises a radiometal chelated by the chelator. In some aspects, the radiometal is226Ac,225Ac,161Tb,155Tb,149Tb,152Tb, or177Lu. In some aspects, a method of delivering a radioisotope or a method of using the in vivo radioisotope targeting construct to deliver the radioisotope to a selected location within the body of a mammalian subject is provided. In some aspects, the radioisotope is used to cause cell death at the selected location within the body.

[0019] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.Brief Description of the Drawings

[0020] 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.

[0021] FIG. 1 shows the decay chain for225Ac.

[0022] FIG. 2 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] FIGs. 3A-3D show the results of concentration-dependent radiolabelling studies of H3TRICA and DOTA with [225Ac]Ac3+(100 kBq) in NH4OAc (1 M, pH 7) (FIG. 3A), [155Tb]Tb3+(100 kBq) in NH4OAc (0.5 M, pH 6) (FIG. 3B), [161Tb]Tb3+(100 kBq) in NH4OAc (0.5M, pH 6) (FIG. 3C), and [177Lu]Lu3+(300 MBq in NH4OAc) (0.5 M, pH 5.5) (FIG. 3D).

[0024] FIGs. 4A and 4B show human serum stability studies of H3TRICA with [225Ac]Ac3+(100 kBq) (FIG. 4A) and [155Tb]Tb3+(300 kBq) (FIG. 4B) monitored for one half-life of the respective radiometal. All reactions were performed at 37 °C and checked with radio-TLC.

[0025] FIGs. 5A, 5B, 5C and 5D show HPLC-MS, UV spectrometry, mass spectrometry and high resolution mass spectrometry results, respectively, for an exemplary TRICA-Gly-TATE construct synthesized by the inventors.

[0026] FIG. 6 shows concentration-dependent radiolabeling of the exemplary TRICA-Gly- TATE construct with [225Ac]Ac3+in NH4OAC (1 M, pH 7) at ambient temperature (20 °C).Description

[0027] 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.

[0028] 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.

[0029] 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 binding fragment 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, 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.

[0030] 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.

[0031] 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.

[0032] The inventors have now determined that chelators having the general structure (101) can coordinate radioisotopes including225Ac and 161 / 155 / 149 / 152-1-bunder 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.

[0033] The structure (101) is 2,2',2"-(1 ,7,13-trioxa-4,10,16-triazacyclooctadecane-4,10,16- triy l)triacetic acid, which is referred to herein as “TRICA”. The inventors have demonstrated that TRICA is a novel effective chelator for large metals such as actinium or terbium which can coordinate under mild conditions and produce a stable complex in vivo. Also, the binding affinity of TRICA for the desired large radiometals such as actinium and terbium is high relative to currently available chelators, allowing the preparation of an in vivo radioisotope targeting chelate construct having a high specific activity. The preparation of in vivo radioisotope targeting chelate constructs having high specific activity may be particularly important for treatment or prophylaxis of conditions in which the target molecule is expressed at relatively low levels, making the target molecule readily saturable in vivo.

[0034] In some embodiments, TRICA can be directly coupled to a biological targeting moiety, optionally with a linker interposing TRICA and the biological targeting moiety, by coupling the biological targeting moiety or linker directly to one of the carboxyl groups of structure (101), e.g. as shown below in structure (102), (103) or (104):R is any arm that allows selective conjugations to biomolecule.(102) (103)Alkylation of one acetate arm to allow conjugation with biomolecule via R(104) or by altering the macrocycle to attach a linker in any suitable manner, for example through one of the modifications described below.

[0035] Alteration of the macrocycle to attach a linker or alkylation of one of the acetate arms of TRICA may be preferable in some embodiments as the coordination and binding properties of TRICA would be preserved in such a structure (i.e. the same coordination number and geometry would be preserved), whereas utilizing an approach exemplified by structure (102) above in which the bifunctional molecule is created by conjugating the TRICA to a biological targeting moiety through one of the acetate arms will result in the loss of one coordinating acetate.

[0036] Furthermore, in some embodiments, one or more of the oxygen atoms of one or more of the carboxyl groups of TRICA may be independently substituted by a different heterotatom, e.g. N or S.

[0037] In more detail, in some embodiments, a bifunctional chelator is provided by replacing one of the acetate arms of the TRICA chelator as shown below as (105). In one embodiment, one of the acetate arms is modified with a moiety corresponding to any armthat allows selective conjugation of the chelator to a linker and / or biomolecule that can be used as a targeting agent. In some embodiments, Ri is a benzyl group with any reactive moiety attached, or a picolinate group with any reactive moiety attached, or a primary amine attached to an alkyl linker (n= 1 , 2, 3, 4, or 5) or any linker with compatible reactivity.

[0038] For example, in some embodiments, a bifunctional chelator is provided having structure (106), wherein each R2 is independently, , OH or an amine such aswherein n is 1 , 2, 3, 4 or 5, and wherein if one R2 is OH or an amine group, then the other two R2 are H.For example in some embodiments the bifunctional chelator has one of the structures (107)- (111) shown below.

[0039] In some embodiments, the bifunctional chelator has the structure (112) shown below:

[0040] In some embodiments, a bifunctional chelator is provided by conjugating the linker and / or biomolecule through one of the acetate arms of the TRICA chelator as shown below as (113) via modification of one of the acetate arms to provide an amide linkage. In some embodiments, Ri is a linker and / or a biological targeting moiety as described herein. For example, the acetate can be modified to an activated ester with N-hydroxysuccinimide (NHS) attached as shown below as (114), which is then reacted with a linker or biotargeting molecule to achieve (103).(113) (114)

[0041] In some embodiments, a bifunctional chelator is provided by modifying one of the acetate arms of the TRICA chelator by alkylation as shown below as (115). In one embodiment, one of the acetate arms is modified with an alkyl moiety that allows selective conjugation of the chelator to a linker and / or biomolecule that can be used as a targeting agent. For example, in some embodiments, Ri is a propionic acid with suitable protecting groups or the anhydride version (propionic anhydride); Ri is a butyric acid with suitable protecting groups; Ri is an alkyl amine (n = 1 , 2, 3); or Ri is a benzyl with a reactive functional group (e.g., amine, hydroxyl, isothiocyanate) at the para, ortho or meta position.For example, in some embodiments a bifunctional chelator has one of the following structures:

[0042] In some embodiments, rather than modifying the acetate arms of the TRICA chelator to allow for bifunctionalization, the macrocycle is modified, for example by alkylation or by the introduction of an aromatic ring, to attach a linker and / or biological targeting moiety to the chelator.

[0043] For example, in some embodiments, alkylation of the carbon closest to one of the oxygen atoms in the macrocycle is carried out, as shown below as (121) wherein the star indicates chirality, i.e. R or S, if Ri is not H. In some embodiments, each Ri is independently H or an alkyl amine (n = 1 , 2, 3, 4). In some embodiments, Ri is a benzyl group with a reactive functional group (e.g., amine, hydroxyl, isothiocyanate) attached to the benzyl at the para, ortho or meta position.For example, in some embodiments, the bifunctional TRICA chelator has one of the following structures:(122) (123)

[0044] In some other embodiments, alkylation of the carbon closest to one of the nitrogen atoms in the macrocycle is carried out, as shown below as (124) wherein the star indicates chirality, i.e. R or S. In some embodiments, each Ri is independently H or an alkyl amine (n = 1 , 2, 3, 4). In some embodiments, Ri is a benzyl group with a reactive functional group (e.g., amine, hydroxyl, isothiocyanate) attached to the benzyl at the para, ortho or meta position.For example, in some embodiments the bifunctional TRICA chelator has one of the following structures:(125) (126)

[0045] In some embodiments, introduction of an aromatic ring into the macrocycle is carried out to attach a linker and / or biological targeting moiety to the TRICA chelator, for exampleas shown below as (127). In some embodiments, R3 is an hydroxyl group or an amine group and R2 is C, S or N.

[0046] While the above-described modifications to the structure of the TRICA chelator to yield a bifunctional molecule have been described with reference to making only a single modification of the molecule, those skilled in the art will understand that the various modifications described above could be combined in any different combination if for any reason it was desired to do so. For example, in some cases it may be desirable to attach two or three targeting agents to a single chelator, whether the same targeting agent or different targeting agents, and literature examples of triple substituted macrocycles exist. As one example only, three alkylated acetate arms could be provided on the TRICA chelator to maintain the same coordination number but allow for attachment of three targeting vectors. Or in some embodiments trifunctionality of the construct can be achieved, for example by adding a fluorine (F-18) to the targeting agent, e.g. as an engineered peptide, wherein the F-18 allows for imaging or for double imaging where the construct itself is otherwise suitable for imaging.

[0047] In some embodiments as shown in FIG. 2, 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 vivo targeting 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.

[0048] 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.

[0049] 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.

[0050] 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 1.17Some 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 some embodiments, 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 TRICA 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.

[0051] 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);• 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., Barnaski et al. and Kuo et al.18’19 20 21A person skilled in the art could develop and optimize a suitable linker for a particular application and all such linkers are contemplated in various embodiments.

[0052] 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.

[0053] 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 room temperature, 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.

[0054] In some embodiments, the radionuclide 128 or construct 130 is combined with chelator 126 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.

[0055] 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.

[0056] 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.

[0057] In some embodiments, in vivo radioisotope targeting chelate construct 120 is present in mammalian serum, optionally in human serum. In some embodiments, in vivo radioisotope 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 vivoradioisotope targeting chelate construct 120 is present in a mammalian cell, optionally a human cell.

[0058] 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. In some embodiments, the cells that are killed at the selected location are cancer cells. In some embodiments, the radiation is alpha radiation; beta or gamma radiation could be used in other embodiments.

[0059] 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.

[0060] 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 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 someembodiments, 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.

[0061] 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.

[0062] 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 alpha therapy.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] In some embodiments, the metals that can be used as metal 128 include actinides or lanthanides. In some embodiments, the metal is a radioisotope. In some embodiments, the metals that can be used as metal 128 include|n someembodiments, the metals that can beused as metal 128 includemetals that can be used asmetal 128 include

[0068] Without being bound by theory, TRICA is anticipated to exhibit compatibility with a diverse set of lanthanide elements, includingThiSexpected binding compatibility is grounded inTRICA's successful complexation with225Ac, an actinide, which shares coordination chemistry similarities with La3+, as well aswich is situated midway through the lanthanide series and characterized by an ionic radius of 92 pm - and the smallest lanthanide,177Lu, with an ionic radius of 86 pm. Despite variations in ionic radius, the foregoing lanthanides exhibit analogous coordination chemistry, emphasizing TRICA's potential efficacy.

[0069] In some embodiments, TRICA 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 TRICA 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 some embodiments, the metal chelate or the chelating ligand is in its free acid form. In some embodiments, the metal chelate or the chelating ligand is in a partially protonated state.

[0070] 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 some embodiments, 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.

[0071] In one specific example embodiment, the targeting moiety 122 is octreotate. In one example embodiment, a specific composition of octreotate with a TRICA chelator, TRICA- Gly-TATE is provided having structure (138) shown below. Such an embodiment may be of interest, for example, in Sst2r-expressing cancers. Octreotate is a peptide that can be conjugated to chelators that will enable complexation with radiotoxic metals; the peptide iscovalently conjugated to a chelator and this is a precursor that is then complexed with a radiotoxic metal e.g. any of the metals described above, including in some embodiments177Lu,225Ac,161Tb,212Pb,229Th etc. The radiometallated peptide is then injected into a patient for targeted radiotherapy (TRT) or also referred to as targeted alpha therapy (TAT) when using a radioisotope that emits alpha particles. This has been reviewed extensively over the past decade. An exemplary review is found in Lepareur et al.24The inventors have made the exemplary structure shown as (138) below and believe that it will show superior chelating properties for working with alpha and beta-emitting therapeutic isotopes including213Bi,223Ac,161Tb,177Lu,197Hg,227Th.Linker arms as well as albumin binders that separate the chelator from the peptide can be added by those skilled in the art.

[0072] Without being bound by theory, the examples described herein demonstrate that TRICA as a chelator has a high binding affinity for binding radiometals, particularly larger radiometals, including the exemplary radiometals225Ac,177Lu, and155Tb. The high binding affinity of TRICA for such exemplary radiometals is demonstrated for example by the ability of TRICA to form coordination complexes with the radiometals quantitatively at room temperature conditions and neutral pH at chelator concentrations as low as 10’5M or 10-6M, as compared with the current gold standard chelator DOTA which requires higherconcentrations on the order of 10'4M and harsher chelation conditions of 90°C for 30 minutes to obtain a similar degree of labelling, which is too harsh for many biological targeting moieties (e.g. antibodies) to withstand. This high binding affinity allowed for example the generation of an in vivo targeting chelate construct incorporating225Ac with a molar activity (specific activity) of 400 MBq / nmol, considerably higher than the parallel preparation previously generated using DOTA which was able to chelate225Ac with a molar activity of only approximately 200 kBq / nmol.22This difference in specific activity allows for the accumulation of a higher uptake of the radiometal in the tumor tissue as compared with other tissues. Thus, in some embodiments, a radioisotope targeting construct incorporating TRICA as a chelator has a specific activity of at least 400 MBq / nmol.

[0073] Without being bound by theory, the significantly higher specific activity of the in vivo targeting chelate construct may be particularly important where the construct is used against a target with relatively low levels of expression in vivo, which means that the target can be readily saturated by in vivo targeting construct molecules that are not bound to the radiometal, thereby blocking effective delivery of the radiometal to its desired locus of administration. Thus, TRICA is expected to be more effective against targets with low levels of expression in vivo where current chelators do not work well for conducting targeted radiotherapy.

[0074] Furthermore, the inventors found that TRICA effectively chelated the desired radiometals with good stability over several days at 37°C in human serum.

[0075] Thus, from the examples described herein, it can be soundly predicted that TRICA can be used as a chelator for the in vivo delivery of radioisotopes 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

[0076] 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 TRICA

[0077] TRICA was synthesized according to Scheme 1 . Briefly, Hstrica was synthesized following the convergent synthesis shown in Scheme 1 , which is based on a previously reported synthesis by Griffin ef a / .15The synthesis begins from alkylating toluene-p- sulfonamide (1) with 2-(2-chloroethoxy) ethan-1-ol; the alcohol groups are then protected with mesyl chlorides (3). Finally, the macrocycle is obtained by the condensation of 2,2'- oxybis(ethylamine) 7 with 3, using caesium carbonate as the base. Although commercially available, 2,2'-oxybis(ethylamine) (6) is an expensive starting material, and instead it can easily be prepared following a procedure from Wharton et al. as outlined in Scheme 1. 6 was reacted with p-toluenesulfonyl chloride to get 7 in excellent yield (94%). Deprotection of the 8 was achieved using a 33% HBr / AcOH aqueous solution yielding the macrocycle as a tri-HBr salt (9). The acetate pendent arms were installed under standard nucleophilic substitution reaction to yield 10. Hstrica (11) was achieved through acid catalyzed ester hydrolysis in 4 M HOI, followed by reverse-phase high-performance liquid chromatography (HPLC). Go-evaporation of 11 with 1 M HCI yielded its HCI salt as a clear solid. The final ligand and all synthetic intermediates were characterized by NMR spectroscopy and mass spectrometry (SI). Elemental analysis of the final ligand was performed to confirm the purity of the isolated product.Scheme 1. Synthesis of H3TRICA.

[0078] Details of triaza- 18-crown-6 macrocycle synthesis and characterization:

[0079] N,N-bis(2-(2-hydroxyethoxy)ethyl)-4-methylbenzenesulfonamide (2). MeCN (60 mL) was added to toluene-p-sulfonamide (5.00 g, 29.20 mmol) and K2CO3 (16.14 g, 0.12 mol). To this mixture 2-chloroethoxy ethanol (8.00 g, 64.20 mmol) was added dropwise over a period of 10 minutes. The reaction mixture was heated to 75 °C and left stirring for 60 h. After this time, the solvent was removed in vacuo and was replaced with DCM (100 mL). The resulting slurry was filtered through Celite and washed with more DCM (2 x 50 mL). The organic fractions were pooled and concentrated to dryness. The resulting viscousyellow oil was purified via silica gel chromatography (CombiFlash automated purification system; A: hexanes, B: EtOAc; 100% A to 100% B) yielding the title compound as a colourless viscous oil (8.11 g, 80%).1H NMR (400 MHz, CDCI3, 298K) 7.72 (2H, d), 7.34 (2H, d), 3.72 (8H, m), 3.57 (4H, t), 3.38(4H, t), 2.45 (3H, s).13C NMR (101 MHz, CDCI3, 298K) 5 143.59, 135.95, 129.76, 127.28, 72.58, 70.12, 61.57, 49.23, 21.53. LR-ESI-MS (MeOH) 370.1 [M+Na]+; calcd for [CI5H25NO6S + Na]+370.1.

[0080] (((Tosylazanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1- diyl)dimethanesulfonate (3). DCM (60 mL) was added to N,N-bis(2-(2- hydroxyethoxy)ethyl)-4-methylbenzenesulfonamide (3.99 g, 11.53 mmol, 1 equiv) and EtsN (14.00 g, 138.38 mmol, 12 equiv) and the solution cooled in ice. Methanesulfonyl chloride (3.56 g, 31.12 mmol, 2.7 equiv) was then added dropwise over a period of 10 minutes. The reaction mixture was allowed to passively warm to RT and stirred for 4 hours. The reaction mixture was then washed with 1 M HCI (200 mL). The aqueous and organic layer were separated, and the organic layer concentrated to dryness in vacuo. The resulting viscous oil was purified via silica gel column chromatography (CombiFlash automated purification system; A: CH2CI2, B:MeOH; 100% A to 5% B) yielding the title compound as a pale-yellow oil (4.63 g, 80%).1H NMR (400 MHz, CDCI3, 298K) 7.72(2H, d), 7.34(2H, d), 4.34(4H, m), 3.72(4H, m), 3.69(4H, t), 3.40(4H, t), 3.07(6H, s), 2.45(3H, s).13C NMR (101 MHz, CDCI3, 298K) 5 136.39, 129.81 , 127.14, 70.21 , 68.96, 68.71 , 48.99, 37.62, 31.58, 21.54. LR-ESI- MS (MeOH) 526.0 [M+Na]+; calcd for [C17H29NO10S3 + Na]+526.09.

[0081] 1-azido-2-(2-azidoethoxy)ethane (5): NaNs (10.30 g, 158 mmol, 3 equiv.) was added to a solution of bis (2-chloroethyl)ether (7.54 g, 52.7 mmol, 1 equiv) and TBAI (1.02 g, 2.70 mmol, 0.05 equiv.) in dry DMF (60 mL). The suspension was then heated at 90 °C for 48 h behind a blast shield. Upon completion, the volatiles were removed in vacuo, and the resulting residue partitioned between diethyl ether (200 mL) and de-ionised H2O (200 mL). The aqueous phase was separated and extracted further with diethyl ether (3 x 200 mL). The combined organics were concentrated to ca. 200 mL and dried over Na2SO4. The mixture was filtered and the solvent removed in vacuo to yield the title product as a paleyellow oil (7.36 g, 90%).1H NMR (400 MHz, CDCI3, 298K) 3.71 (4H, t), 3.44(4H, t).13C NMR (101 MHz, CDCI3, 298K) 5 70.10, 50.77.

[0082] 2,2'-Oxybis(ethan-1-amine) (6). 1-azido-2-(2-azidoethoxy)ethane (4b) (2.50 g, 16.0 mmol, 1 equiv) was dissolved in dry THF (50 mL) and degassed with nitrogen for 10minutes. A solution of triphenylphosphine (10.10 g, 38.4 mmol, 2.4 equiv) in dry THF (50 mL) was added dropwise to the reaction mixture over 30 minutes at 0 °C. The solution was allowed to passively warm to RT and stirred for a further 3 h. De-ionised H2O (400 mL) was added and the resulting solution stirred overnight. Upon completion, the volume was reduced to ca. 100 mL and extracted with DCM (3 x 30 mL). The aqueous phase was evaporated in vacuo to yield the title product as a pale-yellow liquid (1.17 g, 70%).1H NMR (400 MHz, CDCI3, 298K) 3.51 (4H, t), 2.89(4H, t).13C NMR (101 MHz, CDCI3, 298K) 5 73.11 , 41.80.

[0083] N,N'-(oxybis(ethane-2,1-diyl))bis(4-methylbenzenesulfonamide) (7). 2,2'-Oxybis(ethan-1 -amine) (4c) (1.16g, 11.1 mmol, 1 equiv) was dissolved in MeCN (55 mL), EtsN (6.74g, 9.3mL, 66.6 mmol, 6 equiv) was added, and the resulting solution was cooled in ice. To this solution, toluene-p-sulfonyl chloride (4.25g, 22.2 mmol, 2 equiv) was slowly added over 10 minutes. After this time, the mixture was heated to 65 °C and left stirring for 22h. The orange precipitate was quenched with 1 M HCI (85 mL). The organic solvent was removed in vacuo and replaced with DCM (160 mL). The aqueous and organic phases were separated, and the organic fractions was concentrated to dryness. The product was left to recrystalize the title product as white solid crystals (4.40g, 96%).1H NMR (400 MHz, CDCI3, 298K) 7.77 (d, J = 8.4 Hz, 4H), 7.34 (d, J = 7.9 Hz, 4H), 3.41 (dd, J = 5.5, 4.5 Hz, 4H), 3.10 (dd, J = 5.4, 4.5 Hz, 4H), 2.45 (s, 6H).13C NMR (101 MHz, CDCI3, 298K) 5 143.59, 129.80, 127.10, 69.29, 42.78, 21.57. LR-ESI-MS (MeOH) 413.0 [M+H]+, 435.0 [M+Na]+; calcd for [C18H24N2O5S2 + H]+413.1 , [C18H24N2O5S2 + Na]+435.1.

[0084] 4,10,16-Tritosyl-1,7,13-trioxa-4,10,16-triazacyclooctadecane (8). To a suspension of caesium carbonate (7.77 g, 23.84 mmol, 4 equiv) in MeCN (150 mL), N,N'- (oxybis(ethane-2,1-diyl))bis(4-methylbenzenesulfonamide) (4d) (2.46 g, 5.96 mmol, 1 equiv) and (((T osylazanediyl)bis(ethane-2, 1 -diyl))bis(oxy))bis(ethane-2, 1 -diyl) dimethanesulfonate (3) (10.0g, 5.96 mmol, 1 equiv) were added at 0 °C. The reaction mixture was heated to 70 °C and stirred for 60 hours, then the solvent was removed in vacuo, and DCM (130 mL) was added. The mixture was filtered through Celite and washed with DCM (60 mL). The combined organic fractions were dried in vacuo. The crude product was purified via basic alumina chromatography (CombiFlash automated purification system; A: CH2CI2, B: MeOH; 100% A to 5% B). The title compound was isolated as an off-white fluffy solid (2.16g, 50%).1H NMR (400 MHz, CDCI3, 298K) 7.69 (6H, d), 7.32(6H, d), 3.58 (12H, t), 3.35(12H, t),2.45(91-1, s).13C NMR (101 MHz, CDCI3, 298K) 5 129.78, 127.08, 77.23, 70.74, 49.64. LR- ESI-MS (MeOH) 746.3 [M+H]+; calcd for [C33H45N3O9S3 + Na]+746.2.

[0085] 1,7,13-Trioxa-4,10,16-triazacyclooctadecane (9). 5 (2.54 g, 3.50 mmol) and phenol (2.95 g, 31.3 mmol) were dissolved in 33% HBr / AcOH (105 mL) and stirred under N2. The reaction mixture was then heated to 80 °C and left for 48 h in a sealed vial. After this time the reaction mixture was cooled, and the solvent was removed via distillation. Acetone was added to precipitate the HBr salt of 1 ,7,13-Trioxa-4,10,16- triazacyclooctadecane (6). This was then filtered out, washed with cold acetone (3 x 10 mL) and dried under vacuum to yield the tri-HBr salt of 1 ,7,13-Trioxa-4,10,16- triazacyclooctadecane (6) (1.41 g, 80%). The tri-HBr salt of 1 ,7,13-Trioxa-4,10,16- triazacyclooctadecane (6) can be neutralised by dissolving it in de-ionized H2O and adding portions of anionic exchange resin (IRA 400) until the pH of the solution is 5. The mixture was gravity filtered and the water is then removed in vacuo to yield an off-white solid.1H NMR (400 MHz, D2O, 298K) 3.74 (12H, t), 3.27 (12H, t).13C NMR (101 MHz, D2O, 298K) 5 65.31 , 46.84. LR-ESI-MS (H2O) 262.2 [M+H]+; calcd for [C12H27N3O3 + H]+262.2.

[0086] tri-tert-butyl 2,2',2"-(1 ,7, 13-trioxa-4, 10,16-triazacyclooctadecane-4, 10, 16- triyl)triacetate (10). Dry MeCN (30mL) was under N2 to a mixture of triamine-3HBr (0.20g, 0.397mmol, lequiv), Na2COs (0.50g, 4.76mmol, 12 equiv) and KI (0.016, 0.0992mmol, 0.25 equiv). Tertbutyl bromoacetate (0.618g, 3.17 mmol, 8 equiv) was added dropwise at 0°C. The reaction was allowed to reflux for 3.5d at 70°C. The reaction mixture was gravity filtered and washed with MeCN. The solvent was removed in vacuo and the red oil purified via silica gel column chromatography (CombiFlash automated purification system; A: CH2CI2, B: MeOH; 100% A to 5% B) to isolate the title product as a yellow residue (0.190g, 80%).1H NMR (400 MHz, CDCI3, 298K) 3.54 (12H, t), 3.23 (6H, s), 2.81 (12H, t), 1.46 (27H, s).13C NMR (101 MHz, CDCI3, 298K) 5 171.98, 82.04, 81.52, 67.59, 67.19, 54.94, 54.65, 53.51 , 28.15, 28.13, 1.99. LR-ESI-MS (MeOH) 604.3 [M+H]+calcd for [C30H57N3O9 + H]+604.4.

[0087] Hstrica (11). TRICA-pr (0.190, 0.315mmol) was dissolved in 4M HOI (10 mL) and heated to 80°C for 24h. Upon completion, the volatiles were removed in vacuo and the yellow residue was purified via RP-HPLC (A: H2O (0.1 % TFA), B: MeOH, 0-15 min 100% A, 15-30 min 100% A to 100% B, 10mL / min, tR = 10 min). The fractions were combined and co-evaporated with 4M HOI (3 x 1 mL) and lyophilized to produce a clear colorless solid of 4.01 (6H, s), 3.85 (12H,t), 3.59 (12H, t).13C NMR (101 MHz, CDCI3, 298K) 5 169.32, 64.10, 54.82, 54.54. HR-ESI- MS (H2O) 436.2286 [M+H]+; calcd for [C18H33N3O9 + H]+436.2295. Elemental Analysis: found %: C 32.95, N 6.39, H 6.85; calcd for C18H33N3O9.4HCI.4H2O: C 33.09, N 6.43, H 6.94Example 2.0 - Radiolabelling Studies of TRICA

[0088] In the investigation of HsTRICA's radiolabeling capabilities, a range of radioisotopes was systematically examined for their characteristics and applications. [225Ac]Ac3+was chosen for its extended half-life and emission of four alpha particles, making it suitable for targeted alpha-particle therapy (TAT). The study also evaluated HsTRICA’s compatibility with [155Tb]Tb3+and [161Tb]Tb3+, both acknowledged for their roles in imaging and therapy. [155Tb]Tb3+emerges as a compatible imaging isotope for [225Ac]Ac3+. Expanding further, [177Lu]Lu3+, known for its therapeutic potential in cancer treatment, was included. The radiochemical conversions (RCCs), determined by radio-thin layer chromatography (radio- TLC), are illustrated in FIGs. 3A-3D and further confirmed by radio-HPLC.

[0089] To assess HsTRICA's efficacy in incorporating [225Ac]Ac3+, radiolabeling experiments were conducted at pH 7, using 40-100 kBq of [225Ac]Ac3+. HsTRICA's radiolabeling, performed at 25 °C, showcased quantitative incorporation of [225Ac]Ac3+at 10'6M within 30 minutes, achieving high molar activities of 400 MBq / pmol. Even at a lower concentration (10'7M), H3TRICA retained the ability to incorporate 79% of [225Ac]Ac3+within the same timeframe. In contrast, DOTA, benchmarked in the study, exhibited lower efficiency, incorporating [225Ac]Ac3+only at higher concentrations (10‘5M) and at a higher temperature (85 °C), with radiolabeling efficiency dropping to 35% at 10'6M. The kinetics study revealed a rapid sequestration of [225Ac]Ac3+within the first 5 minutes by H3TRICA, establishing H3TRICA as a highly promising candidate for [225Ac]Ac3+radiopharmaceuticals. In contrast, DOTA requires heating at 80 °C for 30min / 1 h to achieve similar results. Results are shown in FIG. 3A.

[0090] Given potential synergies in isotopic compatibility, concentration-dependent radiolabeling studies of H3TRICA extended to include [155Tb]Tb3+and [161Tb]Tb3+at pH 6 and 25 °C, employing 100 kBq. Notably, H3TRICA quantitatively incorporated both terbium isotopes at 10'5M, followed by a drop to 40% radiolabeling efficiency at 10'6M. Theachievable molar activity with terbium reached 1 GBq / pmol at room temperature, comparable to published chelators like H4noneunpaX. Heated to 80 °C for comparison with DOTA, H3TRICA exhibited an affinity for terbium two orders of magnitude higher than DOTA. Results are shown in FIGs. 3B and 3C.

[0091] Building on observed affinity similarities in solution studies among La3+, Tb3+, and Lu3+, the investigation extended to explore radiolabeling efficacy with [177Lu]Lu3+. Despite promising parallels, H3TRICA exhibited limited radiometal ion compatibility with [177Lu]Lu3+, requiring higher concentrations (10'4M) and an elevated temperature of 80 °C for successful radiolabeling. At a reaction temperature of 37 °C, only a 90% RCC was achieved, likely attributed to the smaller size of Lu3+, suggesting without being bound that H3TRICA is more effective with larger metal ions such as Tb3+and Ac3+. Results are shown in FIG. 3D.

[0092] In more detail, to conduct the above experiments, H3TRICA was dissolved in ultrapure deionized water and then diluted to create a 10'2M stock solution. Subsequent dilutions ranging from 10'3to 10'6M of H3TRICA were prepared. Radiolabeling experiments were conducted in triplicates with a final ligand concentration of 10'3to 10'6M (10 uL) in ultrapure deionized water. Aliquots (5-10 uL) of each radionuclide - [225Ac]Ac(NO3)3 (50-100 kBq) in NH4OAc (1 M, pH 7), [155 / 161Tb]TbCI3(100-1 MBq) in NH4OAc (0.5 M, pH 6), and [177Lu]LuCl3 (300 MBq) in NH4OAc (0.5 M, pH 5.5) - were used. Reactions were incubated at 25°C (37°C, and 80°C for [177Lu]Lu3+), and the RCC % was determined after 30 minutes using radio-TLC with 50 mM EDTA (pH 5.5) as the mobile phase and iTLC-SA plates as the stationary phase. The RCC % of [225Ac]Ac(TRICA) at 10-5M was also determined at 5 and 15 minutes using the same method.

[0093] The foregoing experiment confirms that TRICA exhibits favourable radiolabeling results under mild conditions (room temperature, pH 6-7, 5-30 minutes), which is compatible with biomolecules used as targeting vectors in therapy and diagnostics. TRICA exhibits superior affinity with225Ac compared to crown (as reported by Yang et al - Chem. Eur. J. 2020, 26, 11435-11440), achieving an 80% RCC at 10'7M, while crown's labeling drops to 0%. Additionally, TRICA demonstrates slightly better affinity with225Ac and 155 / ISI-TI-) than noneunpaX (as reported by Wharton et al.3). Specifically, TRICA achieves 80% RCC at 10'7M with225Ac, whereas noneunpaX reaches 70%. In the case of iss / iei-ri-) TRICA isquantitatively radiolabeled at 10'5M, dropping to 60% at 10'6M. Conversely, noneunpaX is radiolabeled at about 90-95% at 10’5M and decreases to 40-50% at 10-6M.Studies with TRICA

[0094] The kinetic inertness of H3TRICA with [225Ac]Ac3+and [155Tb]Tb3+was evaluated by incubation in human serum at 37 °C. Human serum contains endogenous species able to sequester metal ions. Human serum stability studies were performed by addition of human serum (100 uL) to the prepared [225Ac]Ac(TRICA) (100 kBq) and [155Tb]Tb(TRICA) (300 kBq) to get a 200 uL final reaction (n = 3). The reactions were incubated at 37°C for 10 days (for [225Ac]Ac(TRICA)) and 5 days (for [155Tb]Tb(TRICA)) and monitored via radio-TLC.

[0095] Results are shown in FIGs. 4A and 4B. Both radiolabeled complexes demonstrated remarkable stability, maintaining integrity over one radiometal's half-life (10 days for [225Ac]Ac3+and 5 days for [155Tb]Tb3+). Notably, the transchelation rate was found to be less than 5%, showcasing promising potential for in vivo applications.Example 4.0 - Synthesis of Bifunctional TRICA

[0096] The inventors will synthesize bifunctional TRICA having the following structuresBifunctional TRICA (t-butyl) Bifunctional TRICA4-(10, 16-bis(2-(tert-butoxy)-2-oxoethyl)-1 ,7,13-4-(10,16-bis(2-(tert-butoxy)-2-oxoethyl)-1 ,7,13- trioxa-4, 10, 16-triaza cyclooctadeca n-4-yl)-5-(tert-butoxy)-5- trioxa-4, 10, 16-triazacyclooctadecan-4-yl)-5-(tert-butoxy)-5- oxopentanoic acid oxopentanoic acid according to Scheme 2 below:Scheme 2. Proposed synthetic scheme for bifunctional TRICA.

[0097] 5-(benzyloxy)-2-bromo-5-oxopentanoic acid (2). L-Glutamic acid y-benzyl ester (2.5 g, 10.5 mmol) was mixed with KBr (3.75 g, 31.6 mmol) in 1 M HBr (35 mL, 32 mmol) at 0°C. Sodium nitrite (1 .5 g, 21 .05 mmol) was slowly added over 50 min at 0 °C and left stirring for 30 minutes and the reaction was stirred for 24h at ambient temperature. The partitioned solution was extracted with diethyl ether (40 mL x 4). The combined organic fractions were pooled and dried over anhydrous Mg2SO4, and then filtered. The filtrate was concentrated in vacuo and then purified by a silica column (CombiFlash Rf automated column system; 40 g HP silica, A: hexanes B: ethyl acetate, 0-35 % B). The fractions were pooled and the solvent was removed in vacuo to yield a white solid (1.63 g, 51.4%).1H NMR (400 MHz, 298 K, CDCI3): 5 7.39-7.33 (m, 5H), 5.14 (s, 2H), 4.43-4.39 (m, 1 H), 2.64- 2.59 (m, 2H), 2.48-2.39 (m, 1 H), 2.36-2.37 (m, 1 H).

[0098] 5-benzyl 1 -(tert-butyl) 2-bromopentanedioate (3). (2) (1.6 g, 5.3 mmol) in chloroform (10 mL) was added dropwise over 30 min to tert-butyl-2,2,2-trichloroacetimidate(TBTA) (2 mL, 10.9 mmol) in cyclohexane (10 mL). N,N-Dimethylacetamide (1.5 mL, 18 mmol) and boron trifluoride diethyl etherate (200|JL, 1.225 mmol) were then added to the reaction mixture and stirred for 5 days. The suspension was then gravity filtered and the filtrate was concentrated in vacuo and then re-dissolved in DCM (20 mL), followed by washing with water (20 mL x 3) in a separating funnel. The organic fractions were concentrated in vacuo and purified via a silica column (CombiFlash Rf automated column system; 40 g HP silica, A: hexanes B: ethyl acetate, 0-15% B). The product fractions were dried in vacuo to yield a colorless oil (1.44 g, 76%).1H NMR (400 MHz, 298 K, CDCI3): 5 7.36 (s, 5H), 5.13 (s, 2H), 4.25 (m, 1 H), 2.59 - 2.52 (m, 2H), 2.43 - 2.18 (m, 2H), 1.47 (s, 9H).

[0099] di-tert-butyl 2,2'-(1 ,7,13-trioxa-4, 10,16-triazacyclooctadecane-4, 10- diyl)diacetate (5). To a suspension of triamine-3HBr (0.1 g, 0.1983 mmol, lequiv) and tertbutyl bromoacetate (54 mg, 0.28 mmol, 1.4 equiv) in dry MeCN (30 mL) was added a solution of DIPEA (59.4 pL, 44.1 mg, 0.341 mmol, 1.72 equiv) in dry MeCN (30 ml) over 1 hour at 60°C. The reaction was allowed to stir for 4 days at 60°C. The reaction mixture was cooled, and gravity filtered. The filtrate was concentrated in vacuo and purified via silica gel column chromatography (CombiFlash automated purification system; A: DCM, B: MeOH; 100% A to 10% B) to isolate the title product as a yellow residue (32 mg, 46%).1H NMR (400 MHz, CDCI3) 5 3.88 (d, J = 5.2 Hz, 4H), 3.53 (dt, J = 10.9, 4.6 Hz, 8H), 3.37 (s, 4H), 3.13 (s, 4H), 2.93 (s, 8H), 1.44 (s, 18H).

[0100] 5-benzyl 1 -(tert-butyl) 2-(10,16-bis(2-(tert-butoxy)-2-oxoethyl)-1,7,13-trioxa- 4,10,16-triazacyclooctadecan-4-yl)pentanedioate (6). Dry MeCN (1.5 mL) was added to a mixture of (5) (32 mg, 65.3 pmol, lequiv), Na2COs (28 mg, 261 pmol, 4 equiv) and KI (3 mg, 16 pmol, 0.25 equiv). (3) (70 mg, 195 pmol, 3 equiv) was added dropwise at reflux. The reaction was allowed to reflux for 4 d at 70°C. The reaction mixture was gravity filtered and washed with MeCN. The solvent was removed in vacuo and the residue was purified via silica gel column chromatography (CombiFlash automated purification system; A: DCM, B: MeOH; 100% A to 5% B) to isolate the title product as a yellow residue (25 mg, 50%).1H NMR (600 MHz, CDCI3) 6 7.40 - 7.24 (m, 1 H), 5.33 - 5.30 (m, 10H), 3.69 (dd, J = 12.6, 7.8 Hz, OH), 3.57 - 3.48 (m, 1 H), 2.87 - 2.72 (m, 1 H).

[0101] 4-( 10, 16-bis(2-(tert-butoxy)-2-oxoethyl)-1 ,7, 13-trioxa-4, 10, 16- triazacyclooctadecan-4-yl)-5-(tert-butoxy)-5-oxopentanoic acid (7). The mixture of (6)(20 mg, 0.026 mmol), 10% Pd / C (10 mg) in MeOH (4 ml) will be stirred under a H2 atmosphere via H2 balloon for 30 minutes. After filtration, the filtrate will be collected and evaporated to yield (7). Example 5.0 - Preparation and Characterization of Exemplary TRICA-Gly-TATE Construct

[0102] The TRICA-Gly-TATE construct having the structure (138) was synthesized according to Schemes 3 and 4 and determined to have an exact mass of 1522.6472 g / mol.1397 nmol of the synthesized compound was purified by HPLC (obtained as the main peak, labelled as 2) and analyzed by mass spectrometry as shown in FIG. 5A. UV spectrometry was conducted as shown in FIG. 5B, mass spectrometry as shown in FIG. 5C and high resolution mass spectrometry as shown in FIG. 5D.Scheme 3. Synthesis of TRICA for Preparation of TRICA-Gly-TATE Construct.Scheme 4. Synthesis of TRICA-Gly-TATE Biological Targeting Construct.Example 6.0 - Radiolabelling Studies with Octadentate TRICA-Gly-TATE Construct

[0103] The concentration-dependent radiolabeling efficiency of octadentate TRICA-Gly-TATE prepared in Example 5.0 was assessed using [225Ac]Ac3+and compared with the non- bifunctional nonadentate TRICA. TRICA-Gly-TATE was dissolved in ultrapure deionized water and diluted to create a 10'3M stock solution. Subsequent dilutions ranging from 10'4to 10’6M were prepared. Radiolabeling experiments were conducted in duplicates with a final ligand concentration of 10’4to 10-6M (10 uL) in ultrapure deionized water. Aliquots (5- 10 uL) of [225AC]AC(NOS)3 (20 kBq) in NH4OAC (1 M, pH 7) were used. Reactions were incubated at 20°C, and the RCC % was determined after 30 minutes using radio-TLC with 50 mM EDTA (pH 5.5) as the mobile phase and iTLC-SA plates as the stationary phase.

[0104] Both constructs exhibited similar behavior, achieving nearly quantitative RCCs within 30 minutes at ambient temperature when the concentration was 10'6M (FIG. 6). These findings indicate that the conjugation to a biological targeting moiety via one carboxylic acid does not compromise the affinity of TRICA-TATE towards [225Ac]Ac3+.Example 7.0 - LoqD7.4 Measurements of TRICA-Gly-TATE Construct

[0105] Lipophilicity of the radiolabeled chelate bioconjugates described in Example 6.0 was evaluated by determining the distribution coefficients between n-octanol and PBS (pH 7.4). Samples of [225Ac]AcTRICA-Gly-TATE (20 pL) were dispensed into a biphasic solution containing n-octanol (600 pL) and PBS (600 pL, pH 7.4), with each experiment repeated thrice (n = 3). The mixtures underwent vortexing for 2 minutes at ambient temperature followed by centrifugation (10 minutes, 3000 rpm). Subsequently, aliquots of n-octanol (200 pL) and PBS (200 pL) were withdrawn for each sample and subjected to gamma spectroscopy measurements. The log D7.4, representing the logarithm of the ratio between the n-octanol and buffer phases, was calculated using the formula: logw[(n-octanol phase) / (buffer phase)]. [225Ac]AcTRICA-Gly-TATE demonstrated hydrophilic properties with a log D7.4 value of -3.45 ± 0.09, supporting the potential utility of this construct for in vivo use.Example 8.0 - Biodistribution Studies of Bifunctional TRICA Coupled to Targeting Moiety

[0106] In future experiments with the chelator TRICA, a systematic approach will be taken for the synthesis and radiolabeling of TRICA-based bioconjugates. The attachment of TRICA to the peptide TATE will be achieved on-resin, following a similar procedure described by Wharton et al.23Purification of TRICA-TATE will involve high-performance liquid chromatography (HPLC). Radiolabeling studies will be conducted using isotope [155Tb]Tb3+or [161Tb]Tb3+to establish optimal labeling conditions. Quality control, employing radio-TLC and analytical radio-HPLC, will assess the stability and purity of the radiolabeled product. Leveraging insights from previous studies revealing kinetic inertness in human serum, good tumor uptake and no free Tb3+are expected. The new TRICA-TATE construct, designed to maintain similar coordination properties, will be subjected to stability studies in human serum and biodistribution studies in tumor-bearing mice, to validate TRICA as an effective chelator for targeted radiopharmaceuticals.

[0107] 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

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Claims

CLAIMS:1 . A chelator having the structure (I) or an in vivo radioisotope targeting construct comprising a biological targeting moiety and a chelator having the structure (I):

2. A chelator or an in vivo radioisotope targeting construct as defined in claim 1 , having the following structure (105) wherein Ri is a benzyl group with any reactive moiety attached, a picolinate group with any reactive moiety attached, or a primary amine attached to an alkyl linker (n= 1 , 2, 3, 4, or 5) or any linker:optionally having one of the structures (106), (107), (108), (109), (110), (111) or (112).

3. A chelator or an in vivo radioisotope targeting construct as defined in claim 1 , having the following structure (113) wherein Ri is a linker and / or a biological targeting moiety4. A chelator or an in vivo radioisotope targeting construct as defined in claim 1 , having the following structure (115) wherein Ri is a propionic acid with suitable protecting groups or propionic anhydride; a butyric acid with suitable protecting groups; an alkyl amine (n = 1 , 2, 3); or a benzyl with a reactive functional group (e.g., amine, hydroxyl, isothiocyanate) at the para, ortho or meta positionoptionally having one of structures (116), (117), (118), (119) or (120).

5. A chelator or an in vivo radioisotope targeting construct, having: one of the following structures (121) or (124) wherein each Ri is independently H or an alkyl amine having n carbon atoms wherein n is an integer between 1 and 4, a benzyl group with a reactive functional group, optionally amine, hydroxyl, or isothiocyanate, attached to the benzyl at the para, ortho or meta position, and * indicates chirality when Ri is not H, optionally having structure (122), (123), (125) or (126)(121) (124); or having the following structure (127) wherein R3 is an hydroxyl group or an amine group and R2 is C, S or N6. A chelator or an in vivo radioisotope targeting construct as defined in any one of claims 1 to 5, 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, or a click chemistry linker.

7. A chelator or an in vivo radioisotope targeting construct as defined in any one of claims 1 to 6, further comprising a radiometal chelated by the chelator.

8. A chelator or an in vivo radioisotope targeting construct as defined in claim 7, wherein the radiometal comprises9. A chelator or an in vivo radioisotope targeting construct as defined in claim 7, wherein the radiometal comprises 149 / 152 / 155 / 1225 / 226^ 177|_U132 / 133 / 134 / 135|_a161 / 166|_|O153gm150£U165 / 166Qy 169 / 165£r149pm142 / 143 / 145pr or169 / 175yb10. A chelator or an in vivo radioisotope targeting construct as defined in claim 7, wherein the radiometal comprises226Ac,225Ac,161Tb,155Tb,149Tb,152Tb, or177Lu.

11. A chelator or an in vivo radioisotope targeting construct as defined in any one of claims 7 to 10, which has a molar activity of at least 400 MBq / nmol.

12. An in vivo radioisotope targeting construct as defined in any one of claims 1 to 11 , 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.

13. An in vivo radioisotope targeting construct as defined in claim 12, 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, or a diabody.

14. An in vivo radioisotope targeting construct as defined in any one of claims 1 to 13, 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 TRICA substituted for DOTA), BAY 864367 peptide (with TRICA-bound ligand label instead of 18F labeling), RM2 peptide (with TRICA substituted for DOTA), SB3 peptide (with TRICA substituted for DOTA), RM26 peptide, BBN-RGD peptide, Aca- BBN peptide, NeoBOMBI peptide (with TRICA 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.

15. An in vivo radioisotope targeting construct as defined in any one of claims 1 to 14, 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 s (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 (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).

16. An in vivo radioisotope targeting construct as defined in any one of claims 1 to 15, wherein the biological targeting moiety comprises octreotate, optionally wherein the construct has the following structure (138):

17. An in vivo radioisotope targeting construct comprising 2,2',2"-(1 ,7,13-trioxa-4,10,16- triazacyclooctadecane-4,10,16-triyl)triacetic acid as a chelator.

18. A pharmaceutical composition comprising an in vivo radioisotope targeting construct as defined in any one of claims 1 to 17 and a pharmaceutically acceptable carrier, excipient or vehicle.

19. 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 7 to 17 bearing the radioisotope to the mammalian subject.

20. A method as defined in claim 19, further comprising allowing the targeting moiety of the in vivo radioisotope targeting construct to enhance the 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.

21. A method as defined in either one of claims 19 or 20, 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 theradioisotope at a temperature of between about 10°C and about 65°C for an incubation period.

22. A method as defined in claim 21 , wherein the temperature is between about 15°C and about 25°C during the incubation period.

23. A method as defined in either one of claims 21 or 22, wherein the incubation period is between about 5 minutes and about 30 minutes.

24. A method as defined in any one of claims 19 to 23, wherein the combining step is carried out at a pH in the range of about 5.0 to about 7.4.

25. A method as defined in any one of claims 19 to 24, wherein the combining step is carried out in aqueous solution that is substantially free of alcohol.

26. A method as defined in any one of claims 19 to 25, further comprising carrying out an imaging procedure to evaluate the localization of the in vivo radioisotope targeting construct within the body, wherein the imaging procedure optionally comprises positron emission tomography (PET) imaging or single-photon emission computerized tomography (SPECT) imaging.

27. A method as defined in any one of claims 19 to 26, 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.

28. A method as defined in claim 27, wherein the in vivo radioisotope targeting construct is used to cause death of cancer cells at the selected location within the body.

29. A method as defined in either one of claims 27 or 28, wherein the radiation comprises alpha radiation.

30. A method as defined in any one of claims 19 to 29, wherein the mammalian subject is a human.31 . A metal chelate comprising a metal and a chelator having the following structure (I):and wherein the metal is selected from the group consisting of: 149 / 152 155 161 -15,32. The metal chelate of claim 31 , wherein the metal225 / 226^o r177|_U33. The metal chelate as defined in either one of claims 31 or 32, wherein the metal is 225Ac[Ac3+],34. The metal chelate as defined in either one of claims 31 or 32, wherein the metal is 161Tb[Tb3+],155Tb[Tb3+],149Tb[Tb3+] or152Tb[Tb3+],35. An aqueous solution comprising the metal chelate as defined in any one of claims 31 to 34.

36. The aqueous solution as defined in claim 35, wherein the aqueous solution is substantially free of alcohol.

37. A method of forming a metal chelate comprising combining a chelator having the structure (I) below with a radiometal in an aqueous solution at a temperature of between 15°C and 25°C38. The method as defined in claim 37, wherein the radiometal iso r39. The method as defined in claim 37, wherein the radiometal is40. The method as defined in any one of claims 37 to 39, wherein the aqueous solution comprises a pH in the range of about 5.0 to about 7.4.41 . A method as defined in any one of claims 37 to 40, wherein said combining step is conducted for a period of between about 5 and about 30 minutes.

42. A method as defined in any one of claims 37 to 41 , wherein the aqueous solution is substantially free of alcohol.

43. A metal chelate as defined in any one of claims 31 to 34 or a metal chelate made by the method of any one of claims 37 to 42 that is present in mammalian serum or mammalian blood, optionally human serum or human blood.

44. A metal chelate as defined in any one of claims 31 to 34 or a metal chelate made by the method of any one of claims 37 to 42 that is present in a mammal, wherein the mammal is optionally a human.

45. A metal chelate as defined in any one of claims 31 to 34 or a metal chelate made by the method of any one of claims 37 to 42 that is present within a mammalian cell, wherein the mammalian cell is optionally a human cell.

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