Multi-arm bifunctional chelators

Abstract

Provided are new multi-arm bifunctional chelators for use in the manufacture of radiolabeled targeting agents for therapeutic or diagnostic use. Also provided are conjugates of the new bifunctional chelators with various cancer antigen targeting agents.

Description

MULTI-ARM BIFUNCTIONAL CHELATORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No.63 / 503,626 filed May 22, 2023, which is hereby incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 22, 2024, is named PT24-020_PCT_ST26.xml and is 205,833 bytes in size. FIELD OF THE INVENTION

[0003] The presently claimed invention relates to the field of bifunctional chelators and linkers. BACKGROUND

[0004] Bifunctional chelators are compounds that include a metal (or metalloid) chelating moiety and a reactive group that can mediate conjugation to a target molecule such as a protein. Dodecane tetraacetic acid (DOTA), IUPAC name 2,2′,2′′,2′′′-(1,4,7,10- Tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid, is a macrocyclic compound capable of chelating radiometals such as90Y,225Ac, and177Lu. Bifunctional DOTA chelators known in the art include, for example, S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (also known as p-SCN-Bn-DOTA) and 1,4,7,10-Tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (also known as DOTA-NHS- ester).

[0005] What is needed and provided by the present invention are new bifunctional chelator compounds that include multiple chelator moieties within each molecule. SUMMARY OF THE INVENTION

[0006] One aspect of the invention provides a multi-arm bifunctional chelator compound including the structure: H N o Formula Iwherein, n is zero or an integer equal to or less than 110 m is zero or an integer equal to or less than 110 each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is a heteroatom, for example, selected from O and S R is C, CH, or N, o is equal to 3 when R is C and o is equal to 2 when R is CH or N, R1includes a chelator moiety, such as any of those disclosed herein, and R2includes a reactive group or an oligo glycine tag, such as any of those disclosed herein. In one variation, each of n and m is independently an integer in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another variation, all X are the same. In another variation, all Y are the same. In one variation, all X are the same. In one variation, all Y are the same. X and Y may, for example, be the same or different. The values or value ranges of n and m may be the same or different. In a further variation, R1includes a 3-arm or 4-arm DOTA chelator moiety. In another variation, R2includes a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group, or an oligo glycine tag, such as (G)3-6such as a hexaglycine tag. R2may, for example include: or

[0007] A related aspect of the invention provides a multi-arm bifunctional chelator compound having the structure:H 2 N YXRn3 Formula II wherein,n zero or an to or m is zero or an integer equal to or less than 110 each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is a heteroatom, for example, selected from O and S R is a pentaerythritol core structure, R1includes a chelator moiety, such as any of those disclosed herein, and R2includes a reactive group or an oligo glycine tag, such as any of those disclosed herein. In one variation, n is an integer in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another variation, all X are the same. In a further variation, R1includes a 3-arm or 4-arm DOTA chelator moiety. In another variation, R2includes a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group, or an oligo glycine tag, such as (G)3-6 such as a hexaglycine tag. R2may, for example include: or

[0008] Another aspect of the invention provides a multi-arm bifunctional chelator compound having the structure:R2H NH N 3 , wherein,n is zero or an integer equal to or greater than 1, such as an integer in the range of 1 to 110, each X is independently CH2, O, N-CH3, or NH, R is a pentaerythritol core structure, R1is a chelator moiety, such as any of those disclosed herein, and R2is a reactive group, such as any of those disclosed herein. In one variation, n is an integer in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another variation, all X are the same. In a further variation, R1includes a 3-arm or 4-arm DOTA chelator moiety. In another variation, R2includes a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group, or an oligo glycine tag, such as (G)3-6 such as a hexaglycine tag.

[0009] Another aspect of the invention provides a bifunctional chelator compound having the structure: R23 ,wherein, n is zero or an integer equal to or greater than 1, such as an integer in the range of 1 to 110 each X is independently CH2, O, N-CH3, or NH, R is a pentaerithritol core structure,R1is a chelator moiety such as any of those described herein, and R2is a reactive group or an oligo glycine tag, such as any of those described herein.

[0010] In one variation, n is an integer in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another variation, all X are the same. In a further variation, R1includes a 3-arm or 4-arm DOTA chelator moiety. In another variation, R2includes a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group, or an oligo glycine, such as tag (G)3-6 such as a hexaglycine tag.

[0011] A related aspect of the invention provides a bifunctional chelator compound having the structure: H H N N OO COOH3wherein n is zero or an integer greater than or equal to 1, such as an integer in the range of 1 to 110, such as in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0012] A further aspect of the invention provides a bifunctional chelator compound having the structure: S COOH N N ,S COOH N N S NNN, orCOOH COOH N N .

[0013] A still further aspect of the invention provides a bifunctional chelator compound, including the structure: R23 ,H N XXn 3 3 3 3 wherein,n is zero or an integer in the range of 1 to 110, m is zero or an integer in the range of 1 to 110, each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is a heteroatom, for example, O or S, R is a pentaerythritol core structure, R1includes a chelator moiety such as any of those disclosed herein, and R2includes a reactive group or an oligo glycine tag, such as any of those disclosed herein. In one variation, each of n and m is independently an integer in the range of 1 to 40, or 1 to 30, or 1 to 20 or 1 to 10, such as n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another variation, all X are the same. In another variation, all Y are the same. In one variation, all X are the same.In one variation, all Y are the same. X and Y may, for example, be the same or different. The values or value ranges of n and m may be the same or different. In a further variation, R1includes a 3-arm or 4-arm DOTA chelator moiety. In another variation, R2includes a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group, or an oligo glycine tag, such as (G)3-6 such as a hexaglycine tag. R2may, for example include: or

[0014] A related aspect of the invention provides a conjugated molecule formed by reacting any of the bifunctional chelator compounds disclosed herein via its reactive group and a molecule such as a peptide, such as a synthetic peptide, or a protein, such as a recombinant protein. For example, one aspect provides a conjugated molecule formed by reacting one of any of the bifunctional chelator compounds disclosed herein that includes a isothiocyanate (abbreviated “SCN” herein) reactive group and a molecule including one or more primary amine groups, for example, a peptide, such as a synthetic peptide, or a protein, such as a recombinant protein.

[0015] The molecules that are conjugated with the bifunctional chelator compounds may, for example, include antibodies such as monoclonal antibodies, or antibody chains, such as immunoglobulin heavy chains and / or immunoglobulin light chains, and / or the variable regions of such heavy or light chains, or soluble receptor proteins or soluble receptor fusion proteins. The molecules that are conjugated with the bifunctional chelator compounds may, for example, include antigen binding fragments of monoclonal antibodies such as Fab fragments or Fab2 fragments, or corresponding scFv molecules.

[0016] Additional features, advantages, and aspects of the invention may be set forth or apparent from consideration of the following detailed description, drawings if any, and claims. Moreover, it is to be understood that both the foregoing summary of the invention and thefollowing detailed description are exemplary and intended to provide further explanation without limiting the scope of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 illustrates the conjugation of a SCN-tetraethyl-DOTA3(4-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0018] FIG. 2 illustrates the conjugation of a SCN-tetra-PEG2-DOTA3(4-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0019] FIG. 3 illustrates a chemical process for the synthesis of a SCN-tetraethyl-DOTA3(4- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0020] FIG. 4 illustrates the conjugation of a SCN-tetraethyl-DOTA3(3-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0021] FIG. 5 illustrates the conjugation of a SCN-tetraethyl-(R)3bifunctional chelator embodiment of the invention to an antibody in which R includes a chelator moiety. R may be any suitable or desired chelator moiety such as any of those disclosed herein, such as DOTA (4-arm), DOTA (3-arm), NOTA, HEPA, HEHA, TETA, or DTPA.

[0022] FIG. 6 illustrates a chemical process for the synthesis of a SCN-tetraethyl-DOTA3 (3- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0023] FIG.7 illustrates a chemical process for the synthesis of a SCN-tetra-PEG2-DOTA3 (4- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0024] FIG. 8 illustrates a chemical process for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is 4-arm DOTA.

[0025] FIG. 9A illustrates the conjugate of an immunoglobulin antibody with a multi-arm bifunctional chelator embodiment of the invention in which group R is or includes a chelator moiety. Each of FIGS. 9B-9F exemplifies a suitable chelator moiety, namely 4-arm DOTA (FIG.9B), NOTA (FIG.9C), Oxo-DO3A (FIG.9D), PCTA (FIG.9E), and HEHA (FIG.9F).

[0026] FIG.10 illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is SCN.

[0027] FIG. 11A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is maleimide. FIG. 11B illustrates the conjugation of the bifunctional chelator of FIG.11A to an antibody.

[0028] FIG. 12A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in whichthe chelator moiety is exemplified by 4-arm DOTA and the reactive group is a hexaglycine tag group for use in sortase-mediated conjugation. FIG. 12B illustrates the conjugation of the bifunctional chelator of FIG.12A to an antibody.

[0029] FIG. 13A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is DBCO. FIG.13B illustrates the conjugation of the bifunctional chelator of FIG.13A to an antibody. DETAILED DESCRIPTION

[0030] This disclosure provides new multi-arm bifunctional chelator compounds for use in the manufacture of radiolabeled targeting agents for therapeutic and / or diagnostic use, methods of conjugation using the bifunctional chelator compounds, compositions including the resulting conjugates, and methods for radiolabeling the resulting conjugates. Advantageously, the multi- arm bifunctional chelators disclosed herein permit high specific activity radiolabeling of protein affinity agents such as antibodies.

[0031] FIG. 1 illustrates the conjugation of a SCN-tetraethyl-DOTA3 (4-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0032] FIG. 2 illustrates the conjugation of a SCN-tetra-PEG2-DOTA3 (4-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0033] FIG. 3 illustrates a chemical process for the synthesis of a SCN-tetraethyl-DOTA3(4- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0034] FIG. 4 illustrates the conjugation of a SCN-tetraethyl-DOTA3(3-arm DOTA configuration) bifunctional chelator embodiment of the invention to an antibody.

[0035] FIG. 5 illustrates the conjugation of a SCN-tetraethyl-(R)3bifunctional chelator embodiment of the invention to an antibody in which R includes a chelator moiety. R may be any suitable or desired chelator moiety such as any of those disclosed herein, such as DOTA (4-arm), DOTA (3-arm), NOTA, HEPA, HEHA, TETA, or DTPA.

[0036] FIG. 6 illustrates a chemical process for the synthesis of a SCN-tetraethyl-DOTA3(3- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0037] FIG.7 illustrates a chemical process for the synthesis of a SCN-tetra-PEG2-DOTA3(4- arm DOTA configuration) bifunctional chelator embodiment of the invention.

[0038] FIG. 8 illustrates a chemical process for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is 4-arm DOTA.

[0039] FIG. 9A illustrates the conjugate of an immunoglobulin antibody with a multi-arm bifunctional chelator embodiment of the invention in which group R is or includes a chelatormoiety. Each of FIGS. 9B-9F exemplifies a suitable chelator moiety, namely 4-arm DOTA (FIG.9B), NOTA (FIG.9C), Oxo-DO3A (FIG.9D), PCTA (FIG.9E), and HEHA (FIG.9F).

[0040] FIG.10 illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA. An exemplary heterobifunctional multi-arm pentaerythritol core structure (4arm PEG, 3arm-Hydroxyl, 1arm-Amine) that may be used in the synthesis is commercially available as Catalog No. A7081-1 from JenKem Technology USA (Plano, Texas USA). Variable n may for example be zero or an integer from 1 to 110, such as from 1 to 50, 1 to 40, 1 to 30, 1 to 20 or 1 to 10, such as n equals 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Corresponding embodiments utilizing other chelator moieties, such as any of those described herein, may also be synthesized in a similar manner.

[0041] FIG. 11A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is maleimide. FIG. 11B illustrates the conjugation of the bifunctional chelator of FIG.11A to an antibody.

[0042] FIG. 12A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is a hexaglycine tag for use in sortase-mediated conjugation. FIG. 12B illustrates the conjugation of the bifunctional chelator of FIG.12A to an antibody.

[0043] FIG. 13A illustrates a chemical process utilizing a heterobifunctional multi-arm core for the synthesis of a multi-arm bifunctional chelator embodiment of the invention in which the chelator moiety is exemplified by 4-arm DOTA and the reactive group is the click chemistry reactive group DBCO. FIG.13B illustrates the conjugation of the bifunctional chelator of FIG. 13A to an antibody.

[0044] The multi-arm bifunctional chelators of the invention may, for example, be used to covalently couple chelator groups to a protein or peptide. Thus, while FIGS.1, 2, 4, 5, 9A-9F, 11B, 12B and 13B exemplify chelator conjugated antibodies, corresponding chelator conjugated protein or peptide embodiments for any protein or peptide are also provided by this disclosure. Such a protein or peptide may, for example, be a cancer cell targeting agent that binds to an antigen / target molecule that is preferentially expressed or overexpressed on mammalian cancer cells such as human cancer cells. The protein may, for example, be an affinity agent such as an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof, a nanobody, or an antibody mimetic protein such as a designed ankyrin repeatproteins (DARPin), or a soluble receptor protein, such as a soluble receptor fusion protein, for example, a fusion protein including a soluble receptor ligand binding sequence fused to a human immunoglobulin Fc region.

[0045] Without limitation, the following enumerated aspects of the invention are provided by this disclosure.

[0046] Aspect 1. Any of the multi-arm bifunctional chelator compounds shown and / or described herein, including without limitation in the appended drawings.

[0047] Aspect 2. The bifunctional chelator compound of aspect 1, further including one or more radionuclides chelated thereby.

[0048] Aspect 3. The bifunctional chelator compound of aspect 2, wherein the one or more radionuclides include one or more of134Ce,43Sc,44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,90Y,166Ho,89Zr,97Ru,105Rh,109Pd,111In,117mSn,149Pm,149Tb,153Sm,177Lu,186Re,188Re,199Au,201Tl,203Pb,212Pb,212Bi,213Bi,225Ac,211At, and227Th.

[0049] Aspect 4. A conjugate of a bifunctional chelator compound according to any one of aspects 1-3 with a molecule.

[0050] Aspect 5. The conjugate of aspect 4, wherein the molecule includes a peptide, a synthetic peptide, a protein, a recombinant protein, an antibody, a monoclonal antibody, an antigen-binding fragment of an antibody such as of a monoclonal antibody, an immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, and immunoglobulin light chain, an immunoglobulin light chain variable region, a nanobody, a scFv molecule, or an antibody mimetic protein.

[0051] Aspect 6. A composition including: a quantity of conjugate according to aspect 4 or 5; and at least one pharmaceutically acceptable excipient.

[0052] Aspect 7. A composition including: a quantity of a conjugate according to aspect 4 or 5, wherein the quantity of the conjugate includes a radiolabeled fraction wherein the chelator moiety chelates one or more radionuclides and a non-radiolabeled fraction wherein the chelator moiety does not chelate a radionuclide.

[0053] Aspect 8. The composition of aspect 6 or 7, wherein the one or more radionuclides includes134Ce,43Sc,44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,90Y,166Ho,89Zr,97Ru,105Rh,109Pd,111In,117mSn,149Pm,149Tb,153Sm,177Lu,186Re,188Re,199Au,201Tl,203Pb,212Pb,212Bi,213Bi,225Ac,211At, or227Th.

[0054] Aspect 9. The composition of aspect 7 or 8, wherein the molecule includes a peptide, a synthetic peptide, a protein, a recombinant protein, an antibody, an antigen-binding fragment of an antibody, an immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, an immunoglobulin light chain, an immunoglobulin light chain variable region, or an antibody mimetic protein.

[0055] Aspect 10. The composition of any one of aspects 7-9, further including at least one pharmaceutically acceptable excipient.

[0056] Aspect 11. A method for manufacturing a chelator-conjugated molecule, including the steps: providing a molecule including at least one primary amine group, providing a bifunctional chelator compound according to any one of aspects 1-3 that includes an NHS or SCN reactive group and reacting the compound with the molecule to conjugate the chelator moiety of the bifunctional chelator compound to the molecule; and / or providing a molecule including at least one free thiol group, providing a bifunctional chelator compound according to any one of aspects 1-3 that includes a phenyloxadiazolyl methylsulfone (PODS) reactive group, and reacting the compound with the molecule to conjugate the chelator moiety of the bifunctional chelator compound to the molecule.

[0057] Aspect 12. The method of aspect 11, including the steps of: providing a molecule including at least one free thiol group, providing a bifunctional chelator compound according to any one of aspects 1-3 that includes a PODS reactive group, and reacting the compound with the molecule to conjugate the chelator moiety of the bifunctional chelator compound to the molecule, wherein the step of providing the molecule including at least one free thiol group includes reducing a disulfide bond to generate the at least one free thiol group of the molecule.

[0058] Aspect 13. The method of aspect 11 or 12, wherein the molecule includes a peptide, a synthetic peptide, or a protein.

[0059] Aspect 14. The method of aspect 11 or 12, wherein the molecule includes an antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, an immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, animmunoglobulin light chain, an immunoglobulin light chain variable region, a nanobody, a scFv molecule, or an antibody mimetic protein.

[0060] Aspect 15. The method of aspect 11 or 12, wherein the molecule includes a peptide ligand of a protein such as a receptor protein.

[0061] Aspect 16. The method of any one of aspects 11-15, further including the step of chelating one or more radionuclides to the chelator moiety of the bifunctional chelator compound, such as before the conjugation to the molecule and / or after the conjugation to the molecule.

[0062] Aspect 17. The method of aspect 16, wherein the one or more radionuclides include one or more of134Ce,43Sc,44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,90Y,166Ho,89Zr,97Ru,105Rh,109Pd,111In,117mSn,149Pm,149Tb,153Sm,177Lu,186Re,188Re,199Au,201Tl,203Pb,212Pb,212Bi,213Bi,225Ac,211At, and227Th.

[0063] Aspect 18. A chelator-conjugated molecule prepared according to the method of any one of aspects 11-17.

[0064] Aspect 19. A composition including: a quantity of a chelator-conjugated molecule prepared according to any one of aspects 11-15; and a quantity of a radionuclide, wherein the quantity of the chelator-conjugated molecule includes a radiolabeled fraction wherein the chelator moiety chelates the radionuclide and a non-radiolabeled fraction wherein the chelator moiety does not chelate any radionuclide.

[0065] Aspect 20. The composition of aspect 19, further including at least one pharmaceutically acceptable excipient.

[0066] Aspect 21. The composition of aspect 19 or 20, wherein the radionuclide is selected from134Ce,43Sc,44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,90Y,166Ho,89Zr,97Ru,105Rh,109Pd,111In,117mSn,149Pm,149Tb,153Sm,177Lu,186Re,188Re,199Au,201Tl,203Pb,212Pb,212Bi,213Bi,225Ac,211At, and227Th.

[0067] The chelator moieties of the multi-arm bifunctional chelators recited in the aforementioned aspects may, for example, be any of the chelator moieties disclosed herein such as, but not limited to, 3-arm and 4-arm DOTA chelator moieties.

[0068] Compositions according to the various aspects of the invention may, for example, be radiopharmaceutical compositions and / or radiodiagnostic (radio-imaging) compositions, which may, for example, further include one or more pharmaceutically acceptable carriers or excipients. Such carriers and excipients are well known in the art. For example, injectabledrug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can include excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). An exemplary formulation may be as substantially described in U.S. Patent No. 10,420,851 or International Pub. No. WO 2017 / 155937, incorporated by reference herein. For example, the formulation may include 0.5% to 5.0% (w / v) of one or more excipients selected from the group consisting of ascorbic acid, polyvinylpyrrolidone (PVP), human serum albumin (HSA), a water-soluble salt of HSA, or any combination thereof. The formulation may, for example, include 0.5-5% ascorbic acid; 0.5-4% polyvinylpyrrolidone (PVP); and a chelator conjugated affinity reagent protein such as an antibody, such as a monoclonal antibody, which may, for example, be radiolabeled (include chelated radionuclide), in 50 mM PBS buffer, pH 7.

[0069] While the multi-arm bifunctional chelator embodiments shown in the figures exemplify compounds with particuar chelator moieties, more generally, the invention also provides corresponding bifunctional chelator compounds in which the chelator moiety includes 1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or a derivative thereof; 1,4,7- triazacyclononane-1,4-diacetic acid (NODA) or a derivative thereof; 1,4,7-triazacyclononane- 1,4,7-triacetic acid (NOTA) or a derivative thereof; 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA) or a derivative thereof; 1,4,7-triazacyclononane, 1-glutaric acid-4,7- diacetic acid (NODAGA) or a derivative thereof; 1,4,7,10-tetraazacyclodecane, 1-glutaric acid- 4,7,10-triacetic acid (DOTAGA) or a derivative thereof; 1,4,8,11-tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid (TETA) or a derivative thereof; 1,4,8,11- tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) or a derivative thereof; diethylene triamine pentaacetic acid (DTPA), its diester, or a derivative thereof; 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A''-DTPA) or a derivative thereof; deforoxamine (DFO) or a derivative thereof; 1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H2dedpa) or a derivative thereof; DADA or a derivative thereof; 1,4,7,10-Tetraazacyclododecane-1,4,7,10- tetra(methylene phosphonic acid) (DOTP) or a derivative thereof; 4-amino-6-[[16-[(6- carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7- yl]methyl]pyridine-2-carboxylic acid (MACROPA-NH2) or a derivative thereof; MACROPA or a derivative thereof; 1,4,7,10-tetrakis(carbamoylmethyl)-l,4,7,10-tetraazacyclododecane (TCMC) or a derivative thereof; {4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl- [1,4,7]triazonan-1-yl}-acetic acid (NETA) or a derivative thereof; Diamsar or a derivative thereof; 1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid (TRAP, PRP9, TRAP-Pr) or a derivative thereof; N,N′-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N′-diacetic acid (H4octapa) or a derivative thereof; N,N′-[1- benzyl-1,2,3-triazole-4-yl]methyl-N,N′-[6-(carboxy)pyridin-2-yl]-1,2-diaminoethane (H2azapa) or a derivative thereof; N,N′′-[[6-(carboxy)pyridin-2-yl]methyl]diethylenetriamine- N,N′,N′′-triacetic acid (H5decapa) or a derivative thereof; N,N′-bis(2-hydroxy-5- sulfobenzyl)ethylenediamine-N,N′-diacetic acid (SHBED) or a derivative thereof; N,N′-bis(2- hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED) or a derivative thereof; 3,6,9,15- tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA) or a derivative thereof; desferrioxamine B (DFO) or a derivative thereof; N,N′-(methylenephosphonate)-N,N′- [6-(methoxycarbonyl)pyridin-2-yl]methyl-1,2-diaminoethane (H6phospa) or a derivative thereof; 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N′′,N′′′,N′′′′,N′′′′′-hexaacetic acid (HEHA) or a derivative thereof; 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N′′,N′′′,N′′′′- pentaacetic acid (PEPA) or a derivative thereof; or 3,4,3-LI(1,2-HOPO) or a derivative thereof.

[0070] Various multi-arm bifunctional chelator compounds exemplified herein include SCN reactive groups for conjugation to molecules including primary amine groups. More generally, the invention also provides corresponding embodiments in which the SCN reactive groups are, instead, a reactive group R which may be any suitable reactive group for conjugation of the bifunctional chelator to a target molecule such as a protein or peptide such as but not limited to NHS, phenyloxadiazolyl methylsulfone (PODS), alkyne, and dibenzocyclooctyne (DBCO) reactive groups. The invention also provides corresponding bifunctional chelator compounds having, instead, a 4-pyridyl reactive group for conjugation to molecules including primary amine groups, such as via the Zincke reaction.

[0071] As used herein, the term “antibody” includes, without limitation, (a) an immunoglobulin molecule including two heavy chains and two light chains and which recognizes an antigen; (b) polyclonal and monoclonal immunoglobulin molecules; (c) monovalent and divalent fragments thereof, such as Fab, di-Fab, as well as scFv, diabodies, minibodies, single domain antibodies (sdAb), and nanobodies (VHH); (d) naturally occurring and non-naturally occurring, such as wholly synthetic antibodies, IgG-Fc-silent, and chimeric; and (e) bi-specific and multi-specific forms thereof. Immunoglobulin molecules may derive from any of the commonly known classes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. Antibodies may be human, humanized or nonhuman. When an aspect of the presently disclosed invention refers to or recites an “antibody,” it is intended as referring to any of the full-length antibodies or fragments thereof disclosed herein, unless explicitly denoted otherwise. Further, wherever in this disclosure specific antibodies aredisclosed, corresponding embodiments directed to antigen binding fragments of such antibodies, such as Fab or Fab2fragments, or corresponding scFv molecules are intended to be provided and disclosed. Similarly, wherever in this disclosure specific antibodies are disclosed, corresponding embodiments directed to full-length antibodies, antigen-binding antibody fragments, such as Fab or Fab2 fragments, or scFv molecules that have the same immunoglobulin heavy chain CDRs and / or immunoglobulin light chain CDRs are also intended to be provided and disclosed. Such corresponding embodiments may, for example, include one or both of the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the recited reference antibody. Antibody heavy chain and light chain complementarity determining regions (CDRs) and regions may, for example, be defined / delineated according to the Kabat or IMGT numbering conventions.

[0072] Single chain Fv molecules (scFv) are single polypeptide chain antibody molecules that include an immunoglobulin light chain variable region domain (VL domain) and an immunoglobulin heavy chain variable region domain (VH domain) joined by a peptide linker (L). An scFv molecule represents either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule or VH-L-VL if the VH domain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and for designing suitable peptide linkers are disclosed, for example, in U.S. Patent Nos.4,704,692 and 4,946,778.

[0073] In one aspect, the invention provides a conjugate of any of the bifunctional chelators disclosed herein and a protein, such as a scFv molecule or a protein including at least one scFv molecule segment, that includes, in consecutive order (from amino to carboxyl terminal end), amino acids 1-120 of an immunoglobulin heavy chain variable region as disclosed herein (for example, where amino acid residue 1 is the first residue of the heavy chain variable region according to the Kabat numbering convention), followed by a linker amino acid sequence, followed by the amino acid sequence of a light chain variable region disclosed herein. In one variation the sequence of either or both of the heavy chain variable region portion and the light chain variable region portion of the scFv molecule is minus 1, 2, 3, 4, or 5 amino acids at one or both ends with respect to the full-length variable region sequence. Such conjugates may be radiolabeled by chelation of a radionuclide to the chelator moiety of the conjugate.

[0074] The scFv linker amino acid sequence may, for example, include any of the amino acid sequences: KISGGGGSGGGGSGGGGSGGGGSGGGGSS (SEQ ID NO:122), SPNSASHSGSAPQTSSAPGSQ (SEQ ID NO:123), (G3S)n where, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as (G3S)4, or(G4S)n where, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as (G4S)5.

[0075] It should be further understood herein that wherever in the sequences of this disclosure an immunoglobulin heavy chain sequence or an immunoglobulin light chain sequence are provided that may include an amino terminal signal peptide sequence, corresponding amino acid sequences without the signal peptide sequences are also intended to be disclosed and that the various aspects of the invention may be embodied with either the versions of the proteins with the signal peptide sequence or corresponding versions without the signal peptide sequences, and in any combination.

[0076] A protein, such as an antibody or fragment thereof or scFv molecule, or an antibody mimetic or a peptide that is conjugated to any of the bifunctional linkers disclosed herein may, for example, specifically bind to target antigens specifically expressed by or overexpressed by various types of cancer cells, such as one or more of any of the following cancer antigens, for example the human forms thereof: CD33, DR5, 5T4 (trophoblast glycoprotein), HER2 (ERBB2; Her2 / neu), HER3, TROP2 (TROP-2, EGP1, EGP-1), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD45, CD171, CD138, CS-1, CLL- 1, GD2, GD3, B-cell maturation antigen (BCMA), Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, fibroblast activation protein (FAP), a Somatostatin receptor, Somatostatin Receptor 2 (SSTR2), Somatostatin Receptor 5 (SSTR5), gastrin-releasing peptide receptor (GRPR), NKG2D ligands (such as MICA, MICB, RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, and RAET1N / ULBP3), LYPD3 (C4.4A), Nectin-4, urokinase plasminogen activator receptor (uPAR), Folate receptor alpha (FOLR1), CUB- domain containing protein 1 (CDCP1), Glypican-3 (GPC3), tenascin, tenascin-C, CEACAM5, Cadherin-3, CCK2R, Neurotensin receptor type 1 (NTSR1), human Kallikrein 2 (hK2), norepinephrine transporter, Integrin alpha-V-beta-6, CD37, CD66, CXCR4, Fibronectin extradomain B (EBD), LAT-1, Carbonic anhydrase IX (CAIX), B7-H3 (a / k / a CD276), Disialoganglioside GD2 Antigen (GD2), calreticulin, phosphatidylserine, GRP78 (BiP), TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor a (IL- l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha (Fra), MUCl, epidermal growth factor receptor (EGFR), EGFRvIII, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, DR5, 5T4, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la,MAGE-A1, legumain, HPV E6,E7, MAGE Al, MAGEA3, MAGEA3 / A6, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, prostein, survivin and telomerase, PCTA-l / Galectin 8, KRAS, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B l, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY- TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, GPA7, IGLL1, FGFR2, FGFR2b, Six-transmembrane epithelial antigen of prostate 1 (STEAP1), MUC17, claudin-18 isoform 2 (CLDN18.2), and Sortilin (Neurotensin receptor-3). Such conjugates radiolabeled by chelation with a radionuclide may be used to treat and / or diagnostically image a cancer in a mammalian subject such as a human patient. Targeting agents that are conjugated with a bifunctional chelator as disclosed herein may also include those that bind antigens expressed by immunosuppressive cells found in or associated with cancers such as CCR8 antigen expressed by tumor associated regulatory T-cells (Tregs) and CD33 antigen expressed by myeloid- derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs).

[0077] One aspect of the invention provides a conjugate of any of the bifunctional chelators disclose herein with a protein, such as an antibody, immunoglobulin heavy chain, immunoglobulin light chain, antibody fragment such as Fab fragment, Fab2 fragment or scFv molecules or fusion proteins, that include any of the amino acid sequences or combinations thereof set forth in SEQ ID NOS:1-139.

[0078] Exemplary DR5 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate, include any one or more of the monoclonal anti-DR5 antibodies mapatumumab, conatumumab, lexatumumab, tigatuzumab, drozitumab, and LBY-135.

[0079] Exemplary 5T4 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include any one or more of the monoclonal anti-5T4 antibodies MED10641, ALG.APV-527, Tb535, H6-DM5, and ZV0508.

[0080] Exemplary HER2 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include the monoclonal antibodies trastuzumab and pertuzumab. The amino acid sequences of the light chain and the heavy chain of Trastuzumab reported by DrugBank Online are: light chain (SEQ ID NO:86) and heavy chain (SEQ ID NO:87). The amino acid sequences of the light chain and the heavychain of Pertuzumab reported by DrugBank Online are: light chain (SEQ ID NO:88) and heavy chain (SEQ ID NO:89). The HER2 targeting agent used may, for example, be the ADC fam- trastuzumab deruxtecan-nxki (Enhertu®).

[0081] Exemplary HER3 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include any one or more of the monoclonal antibodies patritumab, seribantumab, lumretuzumab, elgemtumab, GSK2849330, ISU104 (ISU Abxis Co., Ltd) or any of the anti-HER3 antibodies disclosed in U.S. Patent No.10,413,607 or AV-203 (Aveo Oncology) or any of the anti- HER3 antibodies disclosed in U.S. Patent No.10,494,441; U.S. Patent No.9,828,635 or U.S. Pub. No.20210025006. An exemplary HER3 antibody includes an immunoglobulin heavy chain variable region including a CDRH1 including SEQ ID NO:56, a CDRH2 including SEQ ID NO:57, and a CDRH3 including SEQ ID NO:58, an immunoglobulin light chain variable region including a CDRL1 including SEQ ID NO:59, a CDRL2 including SEQ ID NO:60, and a CDRL3 including SEQ ID NO:61. An exemplary HER3 antibody includes an immunoglobulin heavy chain variable region including SEQ ID NO:62 and / or an immunoglobulin light chain variable region including SEQ ID NO:63. An exemplary HER3 antibody includes an immunoglobulin heavy chain amino acid sequence of SEQ ID NO:64 and / or an immunoglobulin light chain amino acid sequence of SEQ ID NO:65. The HER3 targeting agent used may, for example, be the ADC patritumab deruxtecan. Exemplary HER3 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate also include mAb ISU104 (a / k / a Clone No.442S1; ISU Abxis Co., Ltd., Republic of Korea; see, e.g., Hong et al., A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization. Mol Cancer Ther.2021 Jun;20(6):1142- 1152) and any of the anti-HER3 antibodies disclosed in U.S. Patent No.10,413,607. An exemplary HER3 antibody includes heavy chain CDRs, CDR-H1 including sequence DYDMS (SEQ ID NO:144), CDR-H2 including sequence TIDLDSGSIYYADSVQG (SEQ ID NO:145), CDR-H3 including sequence DLHMGPEGPFDY (SEQ ID NO:146), and light chain CDRs, CDR-L1 including sequence SGSSSNIGSNSVS (SEQ ID NO:147), CDR-L2 including sequence SDNHRPS (SEQ ID NO:148), and CDR- L3 including sequence QGWDTSLSGHV (SEQ ID NO:149). A related exemplary HER3 antibody includes a heavy chain variable region including SEQ ID NO:150 and a light chain variable region including the sequence SEQ ID NO:151.

[0082] Exemplary TROP2 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include the monoclonal antibodies Sacituzumab and Datopotamab, antibodies having one or both of the heavy chain and light chain of said antibodies, antibodies having one or both of the heavy chain variable region and the light chain variable region of said antibodies, and antibodies having one or both of the heavy chain CDRs and the light chain CDRs of said antibodies, or TROP2-binding fragments of any of the aforementioned antibodies, such as Fab fragments, Fab2 fragments, or corresponding scFv molecules. Sacituzumab biosimilar is commercially available as Catalog No. A2175 from BioVision Incorporated (an Abcam company, Waltham, MA, USA). Datopotamab biosimilar is commercially available as Catalog No. PX-TA1653 from ProteoGenix (Schiltigheim, France). The TROP2 targeting agent used may, for example, be the ADC sacituzumab govitecan-hziy (Trodelvy®).

[0083] Exemplary TROP2 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include a monoclonal antibody having a heavy chain SEQ ID NO:66 and / or a light chain SEQ ID NO:71 (reported as the heavy and light chains of Sacituzumab), or an antibody including one or both of the heavy chain variable region (SEQ ID NO:67) or the light chain variable region (SEQ ID NO:72) of said chains, or an antibody including 1, 2, or 3 of the heavy chain CDRs of said heavy chain (CDR H1-3: SEQ ID NOS:68-70 respectively) and / or 1, 2 or 3 of the light chain CDRs of said light chain (CDR L1-3: SEQ ID NOS:73-75 respectively), and any of the anti- human TROP antibodies disclosed in U.S. Patent No. 7,238,785 (such as mAb hRS7), U.S. Patent No. 9,492,566, U.S. Patent No. 10,195,517, or U.S. Patent No. 11,116,846, or an antibody including one or both of the heavy chain and light chain variable regions of said antibodies, or an antibody including a heavy chain including 1, 2 or 3 of the heavy chain CDRs of any of said antibodies and / or a light chain including 1, 2, or 3 of the light chain CDRs of any of said antibodies.

[0084] Exemplary TROP2 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate also include the monoclonal antibodies BAT006, BAT007 and BAT008 (Bio-Thera Solutions, Ltd., China) and other anti-TROP2 monoclonal antibodies disclosed in U.S. Patent No. 11,192,954. An exemplary TROP2 antibody includes a light chain sequence including SEQ ID NO:140 and a heavy chain sequence including SEQ ID NO:141. Another exemplary TROP2 antibody includes a light chain sequence including SEQ ID NO:142 and a heavy chain sequence including SEQ ID NO:143.

[0085] Further exemplary TROP2 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include a monoclonal antibody heavy chain SEQ ID NO:76 and / or a light chain SEQ ID NO:81 (reported as the heavy and light chains of Datopotamab), or an antibody including one or both of the variable region of said heavy chain (SEQ ID NO:77) and the variable region of said light chain (SEQ ID NO:82), or an antibody including 1, 2, or 3 of the heavy chain CDRs of said heavy chain (CDRs 1-3: SEQ ID NOS:78-80 respectively) and / or 1, 2 or 3 of the light chain CDRs of the said light chain (CDR H1-3: SEQ ID NOS:83-85 respectively), and any of the anti-human TROP antibodies disclosed in Int’l Pub. No. WO2015098099 or U.S. Pub. No.20210238303, or an antibody including one or both of the heavy chain and light chain variable regions of said antibodies, or an antibody including a heavy chain including 1, 2 or 3 of the heavy chain CDRs of any of said antibodies and / or a light chain including 1, 2, or 3 of the light chain CDRs of any of said antibodies.

[0086] Exemplary CD33 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include the monoclonal antibodies lintuzumab (HuM195), gemtuzumab, and vadastuximab and CD33-binding antibody fragments thereof such as Fab. Fab2 and scFv. An exemplary anti-CD33 antibody that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate includes the CDRs, CDR-H1 including SEQ ID NO:152, CDR-H2 including SEQ ID NO:153, CDR-H3 including SEQ ID NO:154, CDR-L1 including SEQ ID NO:155, CDR-L2 including SEQ ID NO:156, and CDR-L3 including SEQ ID NO:157.

[0087] Exemplary CD38 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include the monoclonal antibodies daratumumab (Darzalex®, Janssen Biotech, Inc.; heavy chain (SEQ ID NO:124), CDR-H1 (SEQ ID NO:125), CDR-H2 (SEQ ID NO:126), CDR-H3 (SEQ ID NO:127), light chain (SEQ ID NO:128), CDR-L1 (SEQ ID NO: 129), CDR-L2 (SEQ ID NO:130) and CDR- L3 (SEQ ID NO:131)), isatuximab (Sarclisa®, a / k / a SAR650984, Sanofi; reported heavy chain SEQ ID NO:132, CDR-H1 (SEQ ID NO:133), CDR-H2 (SEQ ID NO:134), CDR-H3 (SEQ ID NO:135), reported light chain SEQ ID NO:136, CDR-L1 (SEQ ID NO:137), CDR-L2 (SEQ ID NO:138), CDR-L3 (SEQ ID NO:139)), and MOR202 (MorphoSys AG; U.S. Patent No. 8,088,896), as well as CD38-binding fragments thereof and antibodies having the same CDRs as daratumumab, isatuximab, or MOR202. Further exemplary CD38 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include any of the anti-CD38 monoclonal antibodies disclosed in any ofU.S. Patent No.8,088,896, U.S. Patent No.7,829,673, Int’l Pub. No. WO 2008 / 047242, U.S. Pat. No. 8,153,765 and U.S. Pub. No. 20210171653, CD38-binding fragments of any of said antibodies, and antibodies including the same CDRs as said antibodies.

[0088] Exemplary MUC1 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include hTAB004 (OncoTAb, Inc.) and any of the anti-MUC1 antibodies or antibody fragments disclosed in any of U.S. Pub. No. 20200061216 and U.S. Patent Nos.: 8,518,405; 9,090,698; 9,217,038; 9,546,217; 10,017,580; 10,507,25110,517,966; 10,919,973; 11,136,410; and 11,161,911.

[0089] Exemplary LYPD3 (C4.4A) targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include BAY 1129980 (a / k / a Lupartumab amadotin; Bayer AG, Germany) an Auristatin-based anti- C4.4A (LYPD3) ADC or its antibody component Lupartumab, IgG1mAb GT-002 (Glycotope GmbH, Germany) and any of those disclosed in U.S. Pub. No. 20210309711, 20210238292, 20210164985, 20180031566, 20170158775, or 20150030618, 20120321619, Canadian Patent Application No. CA3124332A1, Taiwan Application No. TW202202521A, or Int’l Pub. No. WO2021260208, WO2007044756, WO2022042690, or WO2020138489.

[0090] 5T4 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include but are not limited to 5T4- binding, single chain, chimeric antibody-superantigen fusion proteins such as C215Fab-SEA (SEQ ID NO:93), 5T4Fab-SEAD227A (SEQ ID NO:94), and 5T4Fab-SEA / E-120 (SEQ ID NO:95). Naptumomab estafenatox is SEQ ID NO:96 (chimeric heavy chain component) non- covalently bound to SEQ ID NO:97 (light chain component). In related detail, the heavy chain includes a 5T4 Fab heavy chain component (corresponding to residues 1 to 222 of SEQ ID NO:95), the SEA / E-120 superantigen (corresponding to residues 226 to 458 of SEQ ID NO:95), and a GGP tripeptide linker (corresponding to residues 223-225 of SEQ ID NO:95) covalently linking the Fab heavy chain and SEA / E-120 components. The light chain includes residues 459 to 672 of SEQ ID NO:95.

[0091] 5T4 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein and radiolabeled for use as a radioconjugate include but are not limited to C215Fab-SEA (SEQ ID NO:93), 5T4Fab-SEAD227A (SEQ ID NO:94), 5T4Fab-SEA / E-120 (SEQ ID NO:95); SEQ ID NO:96, Naptumomab estafenatox reported as SEQ ID NO:96 (heavy chain component) non-covalently associated with SEQ ID NO:97 (light chain component), an antibody including the Fab component of Naptumomab estafenatox, an antibody including the heavy chain component of any one of SEQ ID NOS:93-96 (for example, lacking the enterotoxincomponents thereof), a 5T4-binding antibody including the heavy chain component of any one of SEQ ID NOS:93-96 and an associated light chain component such as the light chain component of SEQ ID NO:97, a 5T4-binding antibody (such as a single- or multi-chain antibody, such as but not limited to a human or humanized IgG, such as IgG1) including a heavy chain component that includes 2 or 3 of the heavy chain CDRs present in any one of SEQ ID NOS:93-96, Naptumomab estafenatox modified by covalent linkage of the heavy chain and light chain components to each other by disulfide bonds (between cysteine residues in the manner of a Fab) and / or by a bifunctional thiol reactive crosslinking agent which may include and introduce a chelating moiety (such as DOTA), any of the chimeric 5T4-binding superantigen fusion proteins of U.S. Patent No.7,615,225 U.S. Patent No.10,314,910 and U.S. Pub. No. 20200101160, any of the preceding 5T4 targeting agents in which the antibody component(s) is / are humanized, any of the preceding in which an amino acid (for example, of a heavy chain and / or of a light chain) is substituted to a cysteine, and any of the preceding further including at least one additional amino acid (for example, in a heavy chain and / or in a light chain thereof) of which at least one is a cysteine and / or at least one is a lysine (for example, one or more additional C-terminal amino acids of which one or more are cysteines and / or of which one or more are lysines).

[0092] Light chain variations that may also be used include but are not limited to SEQ ID NO:99 (SEQ ID NO:97 with C-terminal lysine), SEQ ID NO:100 (SEQ ID NO: 97 with C- terminal cysteine), SEQ ID NO:101 (SEQ ID NO:97 with C-terminal lys-cys), and a light chain or light chain segment including 2 or 3 of the CDRs of the light chain of SEQ ID NO:97 – namely CDR-L1 KASQSVSNDVA (SEQ ID NO:119), CDR-L2 YTSSRYA (SEQ ID NO:120), and CDR-L3 QQDYNSPPT (SEQ ID NO:121).

[0093] Further 5T4 targeting agents that may be conjugated with any of the bifunctional chelators disclosed herein, and optionally radiolabeled, include proteins including or consisting of the following: SEQ ID NO:102 [heavy chain portion only of SEQ ID NO:96]; An immunoglobulin heavy chain or heavy chain segment including 2 or 3 of the heavy chain CDRs found in SEQ ID NO:20, namely CDR-H1 GYYMH (SEQ ID NO:116), CDR-H2 RINPNNGVTLYNQKFKD (SEQ ID NO:117), and CDR-H3 STMITNYVMDY (SEQ ID NO:118); SEQ ID NO:103 [SEQ ID NO:96 including linker, excluding enterotoxin portion]; SEQ ID NO:104 [SEQ ID NO:102 with C-terminal lysine]; SEQ ID NO:105 [SEQ ID NO:103 with C-terminal lysine];SEQ ID NO:106 [SEQ ID NO:102 with C-terminal cysteine]; SEQ ID NO:107 [SEQ ID NO:103 with C-terminal cysteine]; SEQ ID NO:108 [SEQ ID NO:102 with C-terminal lys-cys]; SEQ ID NO:109 [SEQ ID NO:103 with C-terminal lys-cys]; SEQ ID NO:110 [5T4Fab-SEA / E-120 (SEQ ID NO:95) with C-terminal lysine]; SEQ ID NO:111 [SEQ ID NO:96 with C-terminal lysine]; SEQ ID NO:112 [5T4Fab-SEA / E-120 (SEQ ID NO:95) with C-terminal cysteine]; SEQ ID NO:113 [SEQ ID NO:96 with C-terminal cysteine]; SEQ ID NO:114 [5T4Fab-SEA / E-120 (SEQ ID NO:95) with C-terminal lys-cys]; SEQ ID NO:115 [SEQ ID NO:96 with C-terminal lys-cys]; and any of SEQ ID NOS:96, 102-109, 111, 113 and 115 in non-covalent association with any of light chain SEQ ID NOS:97 and 99-101 to form a Fab without covalent cross-linking between chains, or a disulfide-bonded (cross-linked) form of such a Fab, or a form of such a Fab covalently cross-linked by a bifunctional cross-linker that may include a chelator such as DOTA.

[0094] In a related embodiment of the invention the 5T4 targeting agent that is conjugated to any of the bifunctional chelators disclose herein includes an antibody, an antigen-binding antibody fragment, such as Fab or Fab2, or an scFv molecule or a fusion protein, that includes immunoglobulin heavy chain CDRs 1, 2 and 3 as set forth in SEQ ID NOS:116, 117 and 118 respectively, and / or immunoglobulin light chain CDRs 1, 2 and 3 as set forth in SEQ ID NOS:119, 120 and 121 respectively. Such 5T4 targeting agents may further include an enterotoxin portion as disclosed herein, such as any of the modified enterotoxins SEA / E-120 (SEQ ID NO:91), SEAD227A(SEQ ID NO:92), and SEQ ID NO:98.

[0095] Where the 5T4 targeting agent includes a heavy chain and a light chain, one or both of the heavy chain and the light chain may be radiolabeled, for example, by chemical conjugation to any of the bifunctional chelators disclosed herein, and chelation of a radionuclide, such as225Ac or177Lu, by the chelator.

[0096] Example: Preparation of a radiolabeled antibody

[0097] Preparing a chelator-conjugated antibody using an SCN (thiocyanate) reactive group bifunctional chelator: antibody conjugates may be prepared by reacting a concentrated solution of monoclonal antibody with a SCN reactive group multi-arm bifunctional chelator as disclosed herein in bicarbonate or in phosphate buffers at pH between about 8 and about 9 and by incubation at either about 37°C or at room temperature. The molar ratio of antibody to bifunctional chelator may, for example, be 1:10 or 1:20, or from 1:10 to 1:20. The conjugatesmay be purified from excess of the bifunctional chelator by repeated filtration or centrifugation and by gravity size exclusion chromatography (SEC). During the purification process, the bicarbonate or phosphate buffer can be changed to N-2-Hydroxyethylpiperazine-N-2- ethanesulfonic acid (HEPES; Free Acid) or acetate medium. Conjugates may be characterized by size exclusion high performance liquid chromatography (SE-HPLC).

[0098] Radiolabeling the conjugate: A chelator-bearing conjugate may first be prepared according to the aforementioned example and then radiolabeled. Where the chelator moiety includes DOTA, DOTAGA or a DOTA derivative as exemplified herein, one radionuclide that may be chelated is225Ac.

[0099] An exemplary labeling reaction for225Ac is as follows: A reaction including 15μl 0.15M NH4Oac buffer, pH=6.5 and 2 μL (10 μg) chelator-conjugated antibody (5 mg / ml) may be mixed in an Eppendorf reaction tube, and 4μL225Ac (10 μCi) in 0.05 M HCl subsequently added. The contents of the tube may be mixed with a pipette tip and the reaction mixture incubated at 37°C for 90 min with shaking at 100 rpm. At the end of the incubation period, 3 μL of a 1mM DTPA solution may be added to the reaction mixture and incubated at room temperature for 20 min to bind the unreacted225Ac into the225Ac-DTPA complex. Instant thin layer chromatography with 10cm silica gel strip and 10mM EDTA / normal saline mobile phase may be used to determine the radiochemical purity of225Ac-chelator-conjugated antibody through separating225Ac-labeled antibody (225Ac-chelator-conjugated antibody) from free225Ac (225Ac-DTPA). In this system, the radiolabeled antibody stays at the point of application and225Ac-DTPA moves with the solvent front. The strips may be cut in halves and counted in the gamma counter equipped with the multichannel analyzer using channels 72-110 for225Ac to exclude its daughters.

[0100] Purification: An exemplary radiolabeled targeting agent, such as225Ac-chelator- conjugated antibody, may be purified either on PD10 columns pre-blocked with 1% I or on Vivaspin centrifugal concentrators with a 50 kDa MW cut-off with 2 x 1.5 mL washes, 3 min per spin. HPLC analyses of the 225Ac-chelator-conjugated antibody after purification may be conducted using a Waters HPLC system equipped with flow-through Waters UV and Bioscan Radiation detectors, using a TSK3000SW XL column eluted with PBS at pH=7.4 and a flow rate of 1ml / min.

[0101] With respect to any of the bifunctional chelator embodiments disclosed herein that include a benzyl group (Bn) adjacent to DOTA or a different chelator moiety, the invention also provides corresponding embodiments in which the benzyl group is replaced by a cyclohexyl group.

[0102] All patents, published patent applications, and other publications cited herein are hereby incorporated by reference in their entireties.

[0103] Although various aspects of the invention or elements thereof are described in terms of “including” or “comprising” throughout the present disclosure and claims, corresponding aspects or elements thereof described in terms of “consisting essentially of” or “consisting of” are also intended to be disclosed and provided.

[0104] While various specific embodiments have been illustrated and described herein, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Moreover, features described in connection with one aspect of the invention may be used in conjunction with other aspects of the invention, even if not explicitly exemplified in combination within.

Claims

WHAT IS CLAIMED IS:

1. A multi-arm bifunctional chelator compound comprising the structure: H N XXo Formula Iwherein, n is zero or an integer in the range of 1 to 110 m is zero or an integer in the range of 1 to 110 each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is O or S, R is C, CH, or N, o is equal to 3 when R is C and o is equal to 2 when R is CH or N, R1comprises a chelator moiety, and R2comprises a reactive group or an oligo glycine tag.

2. The multi-arm bifunctional chelator compound of claim 1, wherein all X and Y are the same.

3. The multi-arm bifunctional chelator compound of claim 1 or 2, wherein n and m are the same.

4. The multi-arm bifunctional chelator compound of any one of claims 1-3, wherein R2comprises: or. ound of any one of claims 1-4, wherein R2comprises a 3-arm or 4-arm DOTA chelator moiety.

6. A multi-arm bifunctional chelator compound having the structure: H N YX3 Formula IIn is zero or an integer in the range of 1 to 110 m is zero or an integer in the range of 1 to 110 each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is O or S R is a pentaerythritol core structure, R1comprises a chelator moiety, and R2comprises a reactive group or an oligo glycine tag.

7. The multi-arm bifunctional chelator compound of claim 6, wherein all X and Y are the same.

8. The multi-arm bifunctional chelator compound of claim 6 or 7, wherein n and m are the same.

9. The multi-arm bifunctional chelator compound of any one of claims 6-8, wherein R2comprises: or. ound of any one of claims 6-9, wherein R2comprises a 3-arm or 4-arm DOTA chelator moiety.

11. A bifunctional chelator compound comprising the structure: H H N N OO COOH3wherein n is zero or an integer in the range of 1 to 110.

12. A bifunctional chelator compound, comprising the structure: S COOH N N ,S COOH N N N13. The bifunctional chelator compound of claim 11 or 12, further comprising one or more radionuclides chelated thereby.

14. The bifunctional chelator compound of claim 13, wherein the one or more radionuclides comprise one or more of86Y,87Y,90Y,111In,177Lu,89Zr,149Tb,153Sm,212Bi,213Bi,225Ac,211At, and227Th.

15. A conjugate of a bifunctional chelator compound according to any one of claims 1-14 with a molecule.

16. The conjugate of claim 15, wherein the molecule comprises a peptide, a synthetic peptide, a protein, a recombinant protein, an antibody, a monoclonal antibody, an antigen- binding fragment of an antibody, an antigen-binding fragment of a monoclonal antibody, an immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, and immunoglobulin light chain, an immunoglobulin light chain variable region, a nanobody, a scFv molecule, or an antibody mimetic protein.

17. A composition comprising: a quantity of a conjugate according to claim 15 or 16; and at least one pharmaceutically acceptable excipient.

18. A composition comprising: a quantity of a conjugate according to claim 15 or 16, wherein the quantity of the conjugate comprises a radiolabeled fraction wherein the chelator moiety chelates one or more radionuclides and a non-radiolabeled fraction wherein the chelator moiety does not chelate a radionuclide.

19. The composition of claim 17 or 18, further comprising at least one pharmaceutically acceptable excipient.

20. A method for manufacturing a chelator-conjugated molecule, comprising the steps: providing a molecule, providing a bifunctional chelator compound according to any one of claims 1-14, and reacting the compound with the molecule to conjugate the chelator moiety of the bifunctional chelator compound to the molecule.

21. The method of claim 20, wherein the molecule comprises a peptide, a synthetic peptide, a protein, or a recombinant protein.

22. The method of claim 21, wherein the molecule comprises an antibody, a monoclonal antibody, an antigen-binding fragment of an antibody, an antigen-binding fragment of a monoclonal antibody, an immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, an immunoglobulin light chain, an immunoglobulin light chain variable region, a nanobody, a scFv molecule, or an antibody mimetic protein.

23. The method of claim 20, wherein the molecule comprises a peptide ligand of a receptor protein.

24. The method of any one of claims 20-23, further comprising the step of chelating one or more radionuclides to the chelator moiety of the bifunctional chelator compound before and / or after performing the conjugation.

25. The method of claim 24, wherein the one or more radionuclides comprise one or more of86Y,87Y,90Y,111In,177Lu,89Zr,149Tb,153Sm,212Bi,213Bi,225Ac,211At, and227Th.

26. A composition comprising: a quantity of a chelator-conjugated molecule prepared according to any one of claims 20-25; and a quantity of a radionuclide chelated by at least a fraction of the quantity of the chelator- conjugated molecule.

28. The composition of claim 27, further comprising at least one pharmaceutically acceptable excipient.

29. The composition of claim 27 or 28, wherein the radionuclide is selected from86Y,87Y,90Y,111In,177Lu,89Zr,149Tb,153Sm,212Bi,213Bi,225Ac,211At, and227Th.

30. A bifunctional chelator compound, comprising the structure: R2H 3 , wherein,n is zero or an integer in the range of 1 to 110, m is zero or an integer in the range of 1 to 110,each X is independently CH2, O, N-CH3, or NH, each Y is independently CH2, O, N-CH3, or NH, Z is O or S, R is a pentaerythritol core structure, R1comprises a chelator moiety, and R2comprises a reactive group or an oligo glycine tag.

31. The bifunctional chelator compound of claim 30, wherein n is an integer in the range of 1 to 20.

32. The bifunctional chelator compound of claim 31 or 32, wherein all X are the same.

33. The bifunctional chelator compound of any one of claims 30-32, wherein R1comprises a 3-arm or 4-arm DOTA chelator moiety.

34. The bifunctional chelator compound of any one of claims 30-33, wherein R2comprises a reactive group which is a SCN, NHS ester, maleimide, DBCO, or PODS reactive group.

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