Squaramide bi-functional chelators and associated radiopharmaceuticals and methods of use

Abstract

Provided herein are bifunctional chelator compounds comprising a cyclic chelator moiety (e.g., 3,6,9,15- tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-acetic acid (PCTA)) and a linker moiety, wherein the linker moiety comprises a terminal squaramate moiety and a C6-10 aryl ring (e.g., a phenyl ring), a click reactive group (e.g., an alkyne, an azide, or a tetrazine), or both. Also provided are radiopharmaceuticals comprising a targeting moiety (e.g., an antibody), a bifunctional chelating linker having the structure of the bifunctional chelator compounds disclosed herein, and a radioactive isotope. Further disclosed are methods of making the bifunctional chelator compounds and radiopharmaceuticals, and their use in the diagnosis and treatment of diseases (e.g., cancer).

Description

SQUARAMIDE BI-FUNCTIONAL CHELATORS AND ASSOCIATED RADIOPHARMACEUTICALS AND METHODS OF USEFIELD

[0001] The present disclosure in the field of biochemistry, immunology, and medicine relates to antibodies (“Abs”) and other proteins conjugated to imaging or therapeutic radionuclides (e.g., radioisotopes) for the diagnosis or treatment of disease.BACKGROUND

[0002] Radiopharmaceuticals typically contain a radioisotope attached to a targeting moiety or carrier. The radioisotope is carried to the target by the carrier where the radioisotope then decays. This decay emits radiation, which can be detected to visualize the target area via imaging or deliver localized radiation doses for therapy. In the field of oncology, radioisotopes commonly used for diagnostic medical imaging decay by gamma or positron emission and are imaged with Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET) imaging respectively. For treatment of cancer, commonly used therapeutic radioisotopes decay by beta-decay, alpha-decay, or other radioactive decay mechanisms, such as Auger emission or internal conversion, that cause cell death in the targeted disease.

[0003] Radioisotopes can bind to a targeting agent, such as an antibody or peptide, either directly or via a bifunctional chelator, depending on the chosen radioisotope and targeting agent. In some cases, conjugation of the chelator, for example DOTA, to the targeting agent can be achieved by using one of its four coordinating arm carboxylic acid groups, which results in reduced chelation stability since the arm is involved in chelation with the isotope (Frullano et al., Curr Org Synth. 2011 Aug 1; 8(4): 535-565). The addition of a linker to the framework of the chelator enables the optimization of the conjugation of the bifunctional chelator to the targeting agent without impacting the stability of chelation.

[0004] When a bifunctional chelator is necessary or desirable, the selection of the specific bi-functional chelator and radioisotope can significantly impact the biodistribution, stability, and efficacy of the radiopharmaceutical. (Strand PLoS ONE 8(8): e70028 Aug 2013, Davey et al, Molecules 2023, 28, 203, Adv Drug Deliv Rev. 2008 Sep; 60(12): 1347-1370.).

[0005] A need therefore exists for bi-functional chelator-antibody radioisotope constructs having improved properties, such as increased in vivo stability.SUMMARY

[0006] The disclosure comprises bifunctional chelators for use in radiopharmaceuticals comprising a PCTA chelator conjugated to a targeting agent using a phenyl squaramate chemical construct.

[0007] Also provided is a bifunctional chelator for use in radiopharmaceuticals comprising a PCTA chelator conjugated to a targeting agent using a click-phenyl-squaramate chemical structure.

[0008] In further embodiments, the bifunctional chelator PCTA phenyl squaramide and PCTA click phenyl squaramide have an average conjugate to antibody ratio of 5 or less.

[0009] In another embodiment, a uPAR-targeting peptide or antibody is chelated to a radioisotope using the bifunctional chelator PCTA phenyl squaramide (PCTA-Sq) or PCTA click phenyl squaramide (PCTA-Click-Sq) to a radioisotope wherein the radioisotope includes but is not limited to Ac-225, Tb-161, Lu-177, Zr-89, Cu-64, Cu- 67, Sn-177m, and In-111.

[0010] In another embodiment, a radiopharmaceutical composition comprises a radiolabeled uPAR-targeting peptide or antibody, unconjugated “cold” uPAR-targeting peptide or antibody, a radioprotectant, and a pharmaceutically acceptable buffer for injection.

[0011] Further provided is a method of treating a patient comprising administering a -targeted radiopharmaceutical concurrent with, prior to, or following administration of unconjugated “cold” targeting agent. For example, the targeting agent may be an antibody or antigen binding fragment thereof, such as a UPAR specific antibody.

[0012] Also provided are drug products comprising a radiopharmaceutical or pharmaceutical composition disclosed herein, and a non-radioactive molecule specific for the same targeting moiety as the radiopharmaceutical.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1: Whole-Body retention of radiolabeled MNPR-101 (using conjugates DFO-NCS vs DFO‘-Sq vs PCTA-NCS).

[0014] FIG. 2: SPECT imaging after administration of 250mCi of MNPR-101-PCTA-NCS-161Tb.

[0015] FIG. 3: Biodistribution of 1 :2 versus 1 :12 MNPR-101-PCTA conjugates with ln-111 in vivo 5 days post injection.

[0016] FIG. 4: SPECT imaging in HT-29 (colon cancer cell line) xenograft mouse models at 2 hours, 4 days, and 7 days after administration of three antibodies utilizing PCTA-Sq linker with Lu-177: trastuzumab (left panel), sacituzumab (middle panel), labetuzumab (right panel).

[0017] FIG. 5: Change in tumor volume demonstrated by MNPR-101-PCTA-Sq-225Ac in a MIA-PaCa-2 (pancreatic cancer cell line) xenograft mouse model.DETAILED DESCRIPTION

[0018] The present disclosure is directed to radiopharmaceuticals (e.g., antibody radioisotope constructs) with a chelating linker (e.g., a bifunctional chelator) that binds to a radioisotope and links the radioisotope to a targeting moiety (e.g., a monoclonal antibody or mAb, small molecule, or peptide). Radiopharmaceuticals comprising a mAb specific for human uPAR to which is chelated a radioisotope or otherwise bound a radioactive isotope are also described. In some embodiments, the targeting moiety is MNPR-101 or its antigen bindingChelators

[0019] Chelating agents based on 3,6,9, 15-tetraazabicyclo-[9.3.1]pentadeca-1 (15),11 , 13-triene-3,6,9-acetic acid (PCTA) show promise as excellent chelators of imaging and therapeutic radioisotopes such as Cu-64, In- 111, Lu-177, and Ac-225 (Journal of Nuclear Medicine July 2016, 57 (7) 1105-1111 , Nucl Med Biol 2015 Feb;42(2) : 164-70) . In recent studies, Mazar et al. previously described radiopharmaceuticals using ln-111 and Ac-225 chelated to PCTA and conjugated to targeting proteins such as monoclonal antibodies which exhibited superior binding and specific activity compared to similarly formulated radiopharmaceuticals using the widely used DOTA chelator (Mazar et al. in U.S. Patent Application Nos. 17 / 749,574 and 17 / 749,763 which are incorporated herein by reference).

[0020] The chemistry, stoichiometry, and chemical structure used to attach the bifunctional chelator to the targeting agent (e.g. a peptide, monoclonal antibody, antibody fragment, etc.) can also significantly impact the biodistribution and stability of the drug. The potential challenges of using chelators that attach to monoclonal antibodies, antibody fragments, or other proteins using isothiocyanate have been reported (U.S. Provisional Patent Application No. 63 / 570,189, Attny docket 32591 / 70242P, incorporated herein by reference). The isothiocyanate is employed by a number of bifunctional chelators (i.e. p-SCN-Bz-DOTA, -DFO, -PCTA, etc.) to conjugate to a biological moiety for radiolabeling.

[0021] Isothiocyanate can react with exposed amines and thiols on antibodies or peptides and form a stable thiourea bond with the amines of lysine and terminally exposed amines. However, when conjugated to thiols such as those present in cysteine residues, the result is a less stable dithiocarbonate (Petri et al. RSC Adv., 2020; 10: 14928-14936) that may be susceptible to cleavage in vivo.

[0022] The selectivity of phenyl isothiocyanate with amines or thiols is predominately determined by the pH of the reaction solution with higher pH (pH 9.0-11) being desirable for amine reactivity while thiol / cysteinepreference increases at pH 6-8 (Petri et al. RSC Adv., 2020; 10: 14928-14936); however, even at higher pH, some binding to the cysteine still occurs. Variations in the pH during manufacture can additionally lead to unacceptably high levels of weaker cysteine binding. This is undesirable because it has been found with various isothiocyanate derivatives that the cysteine adducts can break apart and bind elsewhere (Karlsson et al. Sci Rep. 2016; 6: 21203), which can cause loss of the conjugated chelator from the antibody or peptide. In radioisotope constructs where a peptide or small molecule carrier is used, this issue with in vivo stability might go undetected since the excretion time of such a drug itself is shorter hence the linker breaking off might be difficult to detect.

[0023] Even when the isothiocyanate reacts with the amine of lysine, the thiourea, which is formed in the reaction, may be more susceptible to radiolysis than other linkers as shown in a study of the bifunctional chelator DFO-NCS by Vizier et al, Bioconjugate Chem. 2024, 35, 633-637. The NCS linkage is also unstable in vivo: when Zr-89-labeled antibodies are administered to rhesus monkeys, only constructs with NCS linkers show early clearance through the bladder (Berg E et al, J Nucl Med. 2020 Mar;61(3):453-460). Because antibodies are not cleared through the renal system but small molecules like DFO-Zr-89 are, the NCS-specific presence of Zr-89 in the bladder indicates that the NCS linkage is unstable in vivo and causes loss of Zr-89 from the targeting antibody.

[0024] Another factor impacting the performance of a radiopharmaceutical is the chelator-to-antibody ratio (CAR), which is a ratio of the number of bifunctional chelators attached to a single antibody. For example, Grunberg et al report a higher CAR of the bifunctional chelator DOTA to antibody - 10 DOTA chelators per antibody vs. either 2 or 6 chelators per antibody - positively influences the desired biodistribution (i.e. higher tumor uptake relative to non-specific uptake) of Lu-177 radiolabeled anti-tumor antibody chCE7agl [Grunberg et al, PLoS ONE 8(4): e60350]. Further, in a study by Guleria et al [Guleria Anti-Cancer Agent ME 2018 18(1) 146- 153], Guleria assessed three different conjugation ratios (50:1 CAR, 10:1 CAR, and 5:1 CAR) and concluded that desired properties such as immunoreactivity was best at low (2:1) CAR while non-specific uptake was best at high (50:1) CAR, and that these properties changed depending on the CAR.PCTA Bi-functional Chelator PCTA-Sguaramide (PCTA-Sg) and PCTA Click Sguaramide (PCTA-Click-Sg)

[0025] Provided herein are radiopharmaceuticals (e.g., antibody radioisotope constructs) comprising a chelator (e.g., PCTA squaramide (PCTA-Sq) or PCTA Click Squaramide (PCTA-Click-Sq)- when squaramate is attached to another amine, it becomes a squaramide) that binds to a radioisotope and links the radioisotope to an antibody (e.g., a mAb) or other targeting moiety. In particular, provided are radiopharmaceuticals comprising a bifunctional chelator PCTA-Squaramide (PCTA-Sq), comprising the chelator PCTA and a squaramide linking group to attach to a target moiety, such as an antibody or peptide, for increased binding stability. Also provided are radiopharmaceuticals comprising a bifunctional chelator PCTA and a squaramide linking group containing click chemistry (PCTA-Click-Sq) to attach to a target moiety, such as an antibody or peptide, for increased binding stability. Processes for preparing, conjugating, and labeling the bifunctional chelates are also disclosed.

[0026] Further provided are bifunctional chelator compounds comprising a chelator moiety and a linker moiety, wherein the linker moiety comprises a terminal squaramate moiety and a Cg-w aryl ring, a click reactive group, or both. In some cases, the chelator moiety is a cyclic chelator moiety. In some cases, the cyclic chelator moiety comprises DOTA or derivatives thereof, NOTA or derivatives thereof, HEHA or derivatives thereof, macropa or derivatives thereof, TETA or derivatives thereof, or SarAr or derivatives thereof. In some cases, the cyclic chelator moiety comprises DO2A, DO3A, DOTA-3py, DOTAGA, NODAGA, NOTP, H2BZmacropa, TETPA, DiAmSar, SarAr-NCS, AmBaSar, or BaBaSar. In some cases, the chelator moiety is an acyclic chelator moiety. In some cases, the acyclic chelator moiety comprises DFO*, DTPA, or DFO.

[0027] Bifunctional chelators disclosed herein have a general structure of Formula (0):metal, such as COOH, phosphonic acid, and half-esters thereof, and each R is independently H or a functional group used to attach PCTA to a phenyl squaramate (for PCTA-Sq) or to attach PCTA to a click chemistry group (for PCTA-Click-Sq), wherein a maximum of one R is other than H. Click chemistry group includes alkynes, cyclooctyne, azides, tetrazine, and others known to those skilled in the art. Nonlimiting examples of R include groups with alkyl, cycloalkyl, alkyl, alkenyl, alkynyl, or a substituted version of any of these groups, amide bonds, single or repeating poly-ethylene glycol units, or products of click chemistry, Michael reactions, or esterifications. A bifunctional chelator of Formula (0) can chelate a metal M, forming a complex of Formula (O’):

[0028] Nonlimiting examples of R for PCTA-Click-Sq include(where the squaramate is attached to 1 or more click chemistry groups).

[0029] In some cases, the bifunctional chelators of the disclosure include those of Formula (0a), (Ob), and

[0031] In some cases, each X1is independently COOH, CONH2, PO(OH)2, or a picolinic acid moiety. In some cases, each X1is independently COOH or PO(OH)2. In some cases, at least one X1is COOH. In some cases, at least one X1is CONH2. In some cases, at least one X1is PO(OH)2. In some cases, at least one X1is a picolinic acid moiety. In some cases, each X1is COOH. In some cases, at least one X1is CONH2. In some cases, each X1is PO(OH)2. In some cases, at least one X1is a picolinic acid moiety. In some cases, the picolinic acid moiety has the structure, wherein * represents the point of attachment to the rest of the molecule.

[0032] In some cases, RNis H. In some cases, RNis Ci-6 alkyl. In some cases, RNis methyl.

[0033] In some cases, R° is methyl or ethyl. In some cases, R° is methyl. In some cases, R° is ethyl.

[0034] In some cases, L is a linker moiety comprising an alkyl spacer, a PEG spacer, or both. In some cases, L is a linker moiety comprising an alkyl spacer. In some cases, L is a linker moiety comprising a PEG spacer. In some cases, L is a linker moiety comprising a CMO aryl ring. In some cases, L is a linker moiety comprising a phenyl ring. In some cases, L is a linker moiety comprising a click reactive group. In some cases, L is a linker moiety comprising a CMO aryl ring and a click reactive group. In some cases, the click reactive group is an alkyne moiety, an azide, or a tetrazine moiety. In some cases, the click reactive group is an alkyne moiety. In some cases, the click reactive group comprises a cyclooctyne moiety or a dibenzocyclooctyne-amine (DBCO- amine) moiety. In some cases, the click reactive group comprises a cyclooctyne moiety. In some cases, the click reactive group comprises a dibenzocyclooctyne-amine (DBCO-amine) moiety. In some cases, the click reactive group is an azide. In some cases, the click reactive group is a tetrazine moiety. In some cases, the click reactive group is a 1 ,2,4,5-tetrazine moiety.

[0035] Non-limiting examples of bifunctional chelator compounds of the disclosure are shown in Table A.Table A

[0036] In compound A4 of Table A, R is any natural amino acid or diastereomer thereof, alkyl, aryl, or PEG groups, click-containing functionalities, and n is an integer from 1-20.

[0037] The squaramate is a cyclobutenedione core that has one vinylogous ester and one vinylogous amide. The ester is the reactive center in which after reacting with an amine on a lysine it forms an amide.

[0038] Importantly, the reactivity of the ester can be tuned by changing what is attached to the vinylogous amide. It has been shown by going from an alkyl to a benzene that the rate of reaction increases by 40-fold (Taylor, 2023), significantly improving the time and efficiency of its manufacturing process.

[0039] The addition of click chemistry to PCTA-Sq (for PCTA-Click-Sq) has several advantages. For example, for radioisotopes which are difficult to chelate with PCTA in the mild conditions required for antibodies, this addition of Click chemistry is advantageous. PCTA with the azide part of click chemistry can be labeled with for example with Zirconium-89 at high temperatures (exceeding 90°C) in an aqueous environment for efficient labeling, followed by clicking with an orthogonally conjugated antibody.

[0040] In some embodiments, PCTA can be substituted with a metal chelator described herein comprising a cyclic or acyclic chelator moiety. In some cases, PCTA can be substituted with a metal chelator described herein comprising a cyclic chelator moiety. Exemplary cyclic chelator moieties include, but are not limited to, DOTA and derivatives (including DO2A, DO3A, DOTA-3py, DOTAGA), NOTA and derivatives (including NODAGA or NOTP), PYTA and derivatives, HOPO and derivatives, HEHA and derivatives, macropa and derivatives (including H2BZmacropa), TETA and derivatives (including TETPA), and SarAr and derivatives (including DiAmSar, SarAr-NCS, AmBaSar, BaBaSar). Nonlimiting examples of suitable cyclic chelator moieties include:(Macropa),(H2BZMacropa).Radioisotopes

[0041] The antibody radioisotope constructs of the disclosure comprise one or more radioisotopes. The term “radioisotopes” as used herein refers to radioisotopes of metals that have excess numbers of either neutrons or protons, giving them excess nuclear energy, and making them unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. During those processes, the radioisotope is said to undergo radioactive decay. This decay emits radiation which can be detected to visualize a target area via imaging or to deliver localized radiation doses for therapeutic applications.

[0043] For therapeutic radioisotopes, one such radioisotope is terbium-161 (161Tb) with an oxidation state of +3.161Tb decays by beta emission (average beta energy of 154 keV) with co-emission of internal conversion and Auger electrons, with the latter two having a relatively short effective range compared to its beta emissions. The use of radioisotope Ac-225 for labeling uPAR targeting antibodies referenced in USSN 17 / 749,574 and USSN 17 / 749,763 has the advantage of being an alpha emitter with a short range, which can deliver high radiation doses to the cancer cells. However, this advantage might limit its effectiveness against larger or deeply located tumors. Terbium-161 as mentioned has the advantage of beta emissions, which have longer range and allow deeper penetration into tumor tissues and more uniform radiation to larger metastases or tumors. The short- range Auger electrons can cause more localized and precise cytotoxicity, which is critical for smaller metastatic lesions including micro metastases (Kong et al., Journal of Nuclear Medicine May 2024, 65 (5) 686-687).161Tbalso has the added advantage of a gamma ray emissions (48.9 keV and 74.5 keV), which is useful in Single Photon Emission Computed Tomography (SPECT) imaging. The half-life of161Tb is approximately 7 days, which is similar to the circulation half-lives of many monoclonal antibodies used in medicine.161Tb isotopes have been used in the radiolabeling and evaluation of mAbs in preclinical models (Grunberg et al, 2014 Eur J Nucl Med Mol Imaging). The final decay product of16Tb is dysprosium-161, a stable non-radioactive isotope. Other non-limiting examples of radioisotopes with a half-life sufficient for antibody labeling include Lu-177, Ac-225, Cu-67, and Sn- 177m.

[0044] Non-limiting examples of other radioisotopes that can be substituted for Tb-161 include but are not limited to Tb-161, Zr-89, Cu-64, In-111, Lu-177, Sn-117m and Ac-225. For example, Ac-225 for alpha therapy treatment, Zr-89 or Tb-155 for PET imaging, In-111 orTb-155 for SPECT imaging, or Tb-161 or Lu-177 for both beta therapy (including Auger emission for Tb-161) and SPECT imaging.Antibody conjugates

[0045] To diagnose or treat disease, the ideal targeting agent should target a protein, carbohydrate, lipid, or other entities expressed or present in the target (e.g. a cancer) but rarely or minimally in normal healthy tissue. In various embodiments, the targeting agent is an antibody, antigen binding fragment thereof or other antibody moiety.

[0046] A targeting agent may comprise an antibody moiety (e.g., a mAb) specific for human urokinase plasminogen activator receptor (uPAR), example MNPR-101, other Abs, antigen binding fragments such as single chain Abs (such as scFv), non-Ab polypeptides and peptides, aptamers, etc., as well as small organic molecules. It is contemplated that the radioconjugate described herein is useful when conjugated to an antibody, fragment thereof for other protein moiety to target the radionuclide to a cell for therapy or diagnosis.

[0047] Antibody or protein targets as described herein include but are not limited those set out below and in Table 1 : CD33, CD20, EGFR, PLAUR (uPAR), CD30, HER2, TNFa, CD38, VEGF-A, PSMA, PD-L1, IL1b, PD-1, RANK Ligand, GD2, C5, SLAMF7, CTLA-4, CCR4, RSV, A4-lntegrin, PDGFRa, IgE, VEGFR2, B. anthrasis, IL6R, IL-12 / 23, FOLR1 (FRa), CEACAM5, GPC3 (Glypican-3), CLDN6, LRRC15, CLDN18 (Claudinl 8.2), AFP, PMEL (gp100), GPA33, HK2, MSLN, SLC34A2, (NaPi2b), DLL3, GCC, CAIX, CA9, SLC44A4, MUC16, IL13RA2 (CD213a2), SDC1 (CD138), EDNRB, F3 (TF), MC1R, GRPR.CA12, TROP2, Ep-CAM, MET, MUC1, Nectin-4, CEACAM6, CD70, gpNMB, ROR1, AXL, FAP, HER3, B7-H3, B7-H4, SSTR2, uPA, HGF, IL13RA2, CA-19-9, P- cadherin, EGFRvlll, CD138, CD56, STEAP1, 5T4, CD71, CD37. In some cases, the antibody or protein target is selected from PLAUR (FOLR1 (FRa), CEACAM5, GPC3 (Glypican-3), CLDN6, LRRC15, CLDN18 (Claudin18.2), AFP, PMEL (gp100), GPA33, HK2, MSLN, SLC34A2,(NaPi2b), DLL3, GCC, CA9, SLC44A4, MUC16, IL13RA2 (CD213a2), SDC1 (CD138), EDNRB, F3 (TF), MC1R, GRPR.CA12, TROP2, and Ep-CAM.

[0048] An illustrative list of targeting species is provided in Table 1. The list includes human or humanized or mouse antibodies, antibody drug conjugates, antibody fragments, small molecules, RNA, etc. which are either approved for being used in treating a human or which have been used in clinical trials or currently undergoingdevelopment. When the species listed comprises a targeting agent conjugated to or combined with another agent, this list refers to both the targeting moiety alone and the combined species.Table 1

[0049] While these mAbs showed utility as “naked” antibodies, the present disclosure focuses on their ability to target diagnostic and therapeutic agents, preferably diagnostic and therapeutic radionuclides (radioconjugates), to cancers expressing a cancer antigen.

[0050] A targeting agent may comprise an antibody moiety (e.g., a mAb) specific for human urokinase plasminogen activator receptor (uPAR), example MNPR-101, other antibodies, antigen binding fragments such as single chain Abs (such as scFv), non-Ab polypeptides and peptides, aptamers, etc., as well as small organic molecules, e.g., that have the property of binding to uPAR without inhibiting the binding of uPA as described in US 2023-0338590 A1 , incorporated herein by reference in its entirety.

[0051] The urokinase plasminogen activator receptor (uPAR) has been demonstrated to be selectively upregulated in tumor cells, as well as tumor-associated endothelial, stromal, and inflammatory cells, in a variety of advanced solid tumor types (Mazar, Clin Cancer Res. 2008; 14: 5649-55). In cancers such as breast, colon, pancreatic, stomach, brain, and ovary, uPAR has been observed both in the tumor and surrounding stroma with expression frequently highest at the invasive front of both primary and secondary lesions.

[0052] A significant body of evidence from in vitro and in vivo studies has established that the urokinase plasminogen activator (uPA) system is central to the process of cancer metastasis, making the uPA system a promising target for diagnostic and therapeutic cancer drug development (Mazar et al., Angiogenesis 1999; 3: 15-32). In addition to uPA, its cell surface receptor (uPAR) is a suitable target for the design and development of cancer therapeutic and diagnostic agents (Mazar, AntiCancer Drugs 2001; 12: 397-400) because uPAR is selectively expressed on metastatic tumor cells and angiogenic endothelial cells but rarely, if at all, on other cells.

[0053] uPAR overexpression is associated with poor prognosis in various cancer types and disease aggressiveness (Mazar, Anti-Cancer Drugs 2001 ; 12(5): 387-400, Mazar Clin Cancer Res. 2008; 14: 5649-55). Elevated uPAR expression is often correlated with metastatic and aggressive disease (de Bock et al., Med Res Rev. 2004; 24(1): 13-39, Mazar et al., Curr Pharm Des. 2011; 17: 1970-8). Further, uPAR is rarely expressed in most adult quiescent tissue; when present, it is usually restricted to tissue resident aberrantly activated macrophages and monocytes, as well as remodeling epithelia, which is typically indicative of inflammation or wound healing. Therefore, targeting uPAR may provide a tumor-selective approach for the treatment of cancer and a promising targeting agent for radiopharmaceuticals.

[0054] Monoclonal antibodies (mAbs) that bind to uPA-uPAR complexes and that inhibit their interaction with downstream targets (such as integrins) have also been developed previously. See: U.S. Pat. 8,101,726 and 8,105,602 which are incorporated by reference in their entirety.

[0055] Peptides that bind to uPAR, including AE105, have also been developed previously. See: U.S. Pat. No. 6,277,818 describing uPAR-targeting cyclic peptide compounds that may be conjugated with a diagnostic label and U.S. Pat. No. 6,514,710 also directed to cyclic peptides having affinity for uPAR.

[0056] Antibodies have a longer half-life in circulation compared to smaller molecules such as peptides and typically accumulate in the target gradually over a period of days, which make a longer half-life radioisotope (e.g., a half-life of a day or more) preferable for chelation. For a theranostic, where a common targeting agent is chelated to one radioisotope for diagnosis (e.g., an imaging agent) and another isotope for treatment, it is desirable to have the same bi-functional chelator for both. A common chelator simplifies the conjugatemanufacturing and reduces the potential for differences in biodistribution between diagnostic and therapeutic constructs. Furthermore, use of different isotopes of the same element for diagnosis and therapy (e.g. PET- imaging isotope Copper-64 for diagnosis and the 0-emitter Copper-67 for therapy) further simplifies the manufacture and ensures identical biodistribution.

[0057] The conjugation of the uPAR-targeting antibody MNPR-101 using p-SCN-Bz-PCTA with imaging isotope ln-111 and therapeutic isotope Ac-225 has been described in disclosed and described by U.S. Patent Application No. 18 / 010,510, incorporated herein by reference in its entirety.

[0058] In the field of magnetic resonance imaging, Port et al has previously described in US Patent Application 10 / 560,830 the use of the chelator PCTA conjugated to a peptide via a squaramide bond for diagnostic magnetic resonance imaging exploiting different magnetic resonance properties of the structure using non-radioactive elements of different atomic mass and conjugation limited to peptides.

[0059] The squaramate is a cyclobutenedione core that has one vinylogous ester and one vinylogous amide. The ester is the reactive center in which after reacting with an amine on a lysine it forms an amide. The reactivity of the ester can be tuned by changing what is attached to the vinylogous amide. It has been shown by going from an alkyl to a benzene that the rate of reaction increases by 40-fold (Taylor, 2023).

[0060] uPAR is known to be involved in cancer metastasis (Noh et al, Theranostics. 2013; 3(7) :487-495) with high expression in the tumor, particularly at the tumor edge, and in small metastatic disease. Because of its role in the primary tumor and small metastases, it is desirable to label the antibody with a therapeutic radioisotope that can be effective against diverse sizes of tumors.

[0061] MNPR-101 is a murine and a humanized mAb that directly binds to uPAR with high affinity and specificity (see, for example, MazarAP et al., Curr Pharm Des. 2011 ;17: 1970-8; Marudamuthu AS et al.. J Biol Chem. 2015; 290:9428-41; and U.S. Pat. 8,101,726). MNPR-101 (previously known as huATN-658) is humanized with 96% human sequence to bind human uPAR. MNPR-101 targets a previously unidentified epitope in uPAR, which has been demonstrated to mimic the CD11b binding site on uPAR (Xu X et al., PLoS One. 2014, 9: e85349). CD11 b expression is a marker of myeloid immune cell activation (Pinsky MR, Contrib Nephrol. 2001, 132:354-66), i.e., that of macrophages and neutrophils and is involved in myeloid cell adhesion and infiltration when it interacts with uPAR on these cells (Gu JM et al., J Cell Physiol. 2005, 204:73-82).

[0062] According to the present disclosure, MNPR-101 (or another uPAR specific mAb such as MNPR-102- previously designated ATN-615) serves as a scaffold to design uPAR-specific antibody radioisotope constructs, only to the alternatively activated myeloid immune cells expressing uPAR while sparing normal cells and tissue that do not express uPAR or express very low levels. MNPR-102 is described in e.g., Li, Y. et al., J Mol Biol 2007, 365(4), pp. 1117-29.Variable (V) Region Amino Acid Sequences of two Preferred mAbs mAb MNPR 101 (formerly ATN-658): Variable region sequences

[0063] The consensus amino acid sequence (single-leter code) of the light chain variable region (VL) and heavy chain variable region (VH) polypeptides of MNPR101 are shown below. The complementarity-determining regions (CDRs) for each variable region are highlighted (italic, bold, underscored!

[0064] MNPR-101 VLProtein (SEQ ID NO:1):1 DIXLTQSPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW LLQRPGQSPK 51 RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYCWQGTHFP 101 LTFGAGTKLE LKL

[0065] MNPR-101 VHProtein (SEQ ID NO:2)1 VQLQESGPEL VKTGASVKIS CKASGYSFTS YYMHWVKQSH GKSLEWIGE / 51 NPYNGGASYN QKIKGRATFT VDTSSRTAYM QFNSLTSEDS AVYYCARS / Y101 GHSVLDYNGQ GTTVTVSTABLE 2: CDRs of MNPR-101 L and H Chains*CDR-L1: first CDR of L chain; CDR-H2: 2ndCDR of H chain, etc. mAb MNPR-102: Variable region sequences

[0066] Amino acid sequence (single-leter code) of the light chain (VL) and heavy chain (VH) variable regions of monoclonal antibody MNPR-102. The complementarity-determining regions (CDRs) for each variable region are highlighted (italic, bold, underscored).

[0067] MNPR-102 VLProtein Sequence (SEQ ID NO:9)1 DIVLTQSPDI TAASLGQKVT ITCSASSSVS YMHWYQQKSG TSPKPWIFE / 51 SKLASGVPAR FSGSGSGTSY SLTISSMEAE DAAIYYCQQW NYPF7FGGGT101 KLEIKR

[0068] MNPR-102 VHProtein Sequence (SEQ ID NO:10)1 VKLQQSGPEV VKPGASVKIS CKASGYSFTN FYIHWVKQRP GQGLEWIG Wl51 FHGSDNTEYN EKFKDKATLT ADTSSSTAYM QLSSLTSEDS AVYFCARIVGP101 HWYFDWIGQG TTVTVSSTABLE 3. CDRs of MNPR-102*CDR-L1: first CDR of L chain; CDR-H2: 2ndCDR of H chain, etc.

[0069] According to the present disclosure, an Ab or mAb, has “essentially the same antigen-binding characteristics” as a reference mAb if It demonstrates a similar specificity profile (e.g., by rank order comparison), and has affinity for the relevant antigen (e.g., uPA-uPAR complex) within 1.5 orders of magnitude, more preferably within one order of magnitude, of the reference Ab.Chimeric / Humanized Antibodies

[0070] Antibodies comprise individual chimeric H and L Ig chains. A chimeric L chain comprises an antigen binding region derived from the L chain of a non-human Ab specific for the target antigen, linked to at least a portion of a human CL region. As used herein, the term “antigen binding region” refers to that portion of an Ab molecule which contains the amino acid residues that interact with an antigen and confer on the Ab its specificity and affinity for the antigen. The Ab region includes the “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding (or “contact’) residues.

[0071] As used herein, the term “chimeric antibody” includes monovalent, divalent or polyvalent Igs. A monovalent chimeric Ab is an HL dimer formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric Ab is tetramer H2L2 formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric Ab can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, termed the p chain).

[0072] The disclosure also provides for “derivatives” of mouse mAbs or the chimeric Abs, which term includes those proteins encoded by truncated or modified genes to yield molecular species functionally resembling the Ig fragments.

[0073] Antibodies, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity, can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears ef al., Proc. Natl. Acad. Sci. USA 72.353-357 (1975). With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the Ig chains are separately recovered and then associated. Alternatively, thehosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled Ig, fragment or derivative.

[0074] In some embodiments, in which the radiopharmaceuticals is an antibody radioisotope construct comprising of chelating linker, the average conjugate to antibody ratio (CAR) is less than 5:1. In some embodiments, the conjugate-to-antibody ratio (CAR) is between 1 and 5. In some embodiments, the conjugate- to-antibody ratio (CAR) is between 1 and 2. In some embodiments, the chelating linker is PCTA-Sq with an average CAR between 1 and 5. In some embodiments, in which the chelating linker is PCTA-Sq, the average CAR is less than 1. In some embodiments, in which the chelating linker is PCTA-Sq, the average CAR is between 0.01 and 1. In some embodiments, in which the chelating linker is PCTA-Sq, the average CAR is between 0.02 and 0.5. In some embodiments, the chelating linker is PCTA-Click with an average CAR between 1 and 5. In some embodiments, the chelating linker is PCTA-NCS with an average CAR between 1 and 5. In some embodiments, the chelating linker is PCTA-Sq, PCTA-Click, or PCTA-NCS with an average CAR of about 1 , about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2. In various embodiments, a bifunctional chelator-to-antibody (CAR) ratio is < 5:1, e.g., less than 2:1, or less than 1 :1. In some embodiments, the CAR is less than 2:1. In some cases, the CAR is less than 1:1. In some embodiments, the conjugate-to-antibody ratio is about 1.5. In other embodiments, the conjugate-to-antibody ratio (CAR) is between 0.01 and 1. In some embodiments, the conjugate-to-antibody ratio is about 1 , about 0.8, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, or about 0.1.

[0075] The number of chelating linkers attached to the antibody moiety can affect the biodistribution of the antibody radioisotope construct. As described above, the number of chelating linkers on the antibody can also affect affinity of antibody with its target. Hence, identifying the optimal CAR for diagnostic and therapeutic applications is critical. To achieve high labeling yield with a lower CAR (e.g., CAR of 5 or less, 2 or less, or 1 or less), the target specific activity of the radiopharmaceutical may need to be lowered. Examples in preparation of various antibody radioisotope constructs using different CAR (high and low relative) and different specific activities and benefits of identifying the best CAR are included below.

[0076] In some cases, provided are Antibody Chelator-Sq conjugates of general structure:

[0077] The chelator moiety can be any chelator moiety disclosed herein, e.g., PCTA, yielding e.g., AntibodyPCTA-Sq conjugates.

[0078] Non-limiting examples of Antibody PCTA-Sq and Antibody PCTA-Click-Sq conjugates of the disclosure are shown in Table B.Table BPharmaceutical and Therapeutic Compositions and Their Administration

[0079] The compounds that may be employed in the pharmaceutical compositions of the disclosure include all of the antibody radioisotope constructs described above, as well as the pharmaceutically acceptable salts of these compounds. Pharmaceutically acceptable acid addition salts of the compounds of the disclosure containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art. Exemplary of the acid addition salts that are included in this disclosure are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.

[0080] Pharmaceutically acceptable base addition salts of antibody radioisotope constructs of the disclosure containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hydroxymethyl)methylamine.

[0081] The antibody radioisotope constructs of the disclosure, as well as the pharmaceutically acceptable salts thereof, may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or injectable preparations. Solid or liquid pharmaceutically acceptable carriers may be employed.

[0082] Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like. Similarly, the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension. A summary of such pharmaceutical compositions may be found, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton Pennsylvania (Gennaro 18th ed. 1990).

[0083] The pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.

[0084] The present disclosure may be used in the diagnosis or treatment of any of a number of animal genera and species, and is equally applicable in the practice of human or veterinary medicine. Thus, the pharmaceutical compositions can be used to treat domestic and commercial animals, including birds and more preferably mammals, as well as humans.

[0085] The term “systemic administration” refers to administration of a composition or agent such as the polypeptide, described herein, in a manner that results in the introduction of the composition into the subject’s circulatory system or otherwise permits its spread throughout the body, such as intravenous (i.v.) injection or infusion. “Regional” administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. Examples include intravaginal, intrapenile, intranasal, intrabronchial (or lung instillation), intracranial, intra-aural or intraocular. The term “local administration” refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, subcutaneous (s.c.) injections, intramuscular (i.m.) injections. One of skill in the art would understand that local administration or regional administration often also result in entry of a composition into thecirculatory system, i.e., so that s.c. or i.m. are also routes for systemic administration. Injectables or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Though the preferred routes of administration are systemic, such as i.v., the pharmaceutical composition may be administered topically or transdermally, e.g., as an ointment, cream or gel; orally; rectally; e.g., as a suppository.

[0086] Other pharmaceutically acceptable carriers for polypeptide compositions of the present disclosure are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active polypeptide is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non- homogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipidic layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and / or other materials of a hydrophobic nature. Those skilled in the art will appreciate other suitable embodiments of the present liposomal formulations.

[0087] Therapeutic compositions may comprise, in addition to the antibody radioisotope constructs, one or more additional drugs, such as DNA-damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, radiosensitizers, CAR T-cell therapies, and chemotherapeutic agents. In fact, pharmaceutical compositions comprising any known therapeutic in combination with the antibody radioisotope constructs disclosed herein are within the scope of the disclosure. The pharmaceutical composition may also comprise one or more other medicaments to treat additional symptoms for which the target patients are at risk, for example, anti-infectives.

[0088] The therapeutic dosage administered is an amount which is therapeutically effective, as is known to or readily ascertainable by those skilled in the art. The dose is also dependent upon the age, health, and weight of the recipient, kind of concurrent treatment(s), if any, the frequency of treatment, and the nature of the effect desired, such as, for example, anti-cancer effect.Methods of Treatment

[0089] The radioisotope constructs described in this disclosure are useful for diagnostic or therapeutics uses against a disease or disorder. The constructs for diagnosis and / or therapy may be the same entity, variations of the same chemical construct with a different isotope of the same radioactive element (e.g., imaging and therapy pairs may include Cu-64 and Cu-67 or Tb-155 and Tb-161), or different entities (e.g. Zr-89 for imaging and Tb- 161 for therapy) depending on the type of radioisotopes used.

[0090] Further provided herein are methods of diagnosing or treating a disease or disorder in a patient in need thereof, comprising administering to the patient a diagnostically or therapeutically effective amount of a radioisotope construct disclosed herein.

[0091] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is one or more of breast cancer, lung cancer, ovarian cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, gastric cancer, pancreatic cancer, colorectal cancer, or sarcoma.

[0092] Also provided herein are methods of treating cancer in a patient in need thereof, comprising (i) diagnosing cancer in a patient by administering to the patient an effective amount of a radiopharmaceutical comprisingA targeting moietyA bi-functional chelator comprising: PCTA and a linker comprising a terminal squaramate for binding to the targeting moiety and at least one of a benzene ring and / or click reactive group; and a radioactive isotope;(ii) diagnosing the patient with cancer if the ratio of radiation measured in a first tissue compared to a second tissue is 1.5:1 or greater, and(iii) administering to the patient a therapeutically-effective amount of a cancer therapeutic. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater, and the second tissue is liver tissue. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater, and the second tissue is skeletal muscle tissue.

[0093] In various embodiments, the diagnosing comprises measuring the level of radiation in a first tissue and a second tissue, measured in the patient 10 minutes to 30 days after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and diagnosing the cancer if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater, and the second tissue is liver tissue. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater, and the second tissue is skeletal muscle tissue.

[0094] In various embodiments, the cancer therapeutic is a chemotherapeutic regimen, radiation therapy, antibody therapy, cell therapy, bone marrow transplant surgery, other known cancer therapy, or a combination thereof.

[0095] In various embodiments, the cancer is metastatic cancer.

[0096] A method of treating a disease includes administering a therapeutically effective amount of a radioisotope. The administration may be given once or may be given in one to six cycles with a cycle duration from four to 52 weeks, e.g., four to ten weeks. The administration may also be fractionated with one portion of the therapeutically effective amount administered first followed within one to three weeks by a second portion of the therapeutically effective amount, and this may be repeated in subsequent cycles. For example, an eight-week cycle of treatment may begin with a fractionated dose (Day 1) followed two weeks later (Day 15) by a second fractionated dose before a new cycle starts ten weeks later (Day 43). Subsequent cycles may have the same, increased, or reduced dosing (for example 50% of the cycle 1 dose is given in cycle 2) and may be given as fractions 1 to 3 weeks apart or as a single dose. For example, for dosing of Lu-177 or Tb-161 chelated to an antibody conjugate such as MNPR-101-PCTA-Sq, the fractionated dosing may be between 10 - 60 mCi / m2per fraction. In another example, for dosing of Ac-225 chelated to an antibody conjugate such as MNPR-101-PCTA- Sq, a single dose may be between 10 - 400 pCi. In other cases, for example when Lu-177 or Tb-161 is chelated to a small molecule, peptide, or other conjugate that clears primarily through the kidneys, administered activity per dose may be between 25 - 1000 mCi. In another example, when Ac-225 is chelated to a small molecule, peptide, or other conjugate that clears primarily through the kidneys, administered activity per dose may be between 25-2000 |_iCi.

[0097] In various embodiments, the antibody radioisotope construct comprises: a radioisotope; a chelating linker; and a targeting agent such as an antibody, antibody fragment, or peptide moiety specific to the human urokinase plasminogen activator receptor (uPAR), wherein the chelating linker comprises one or more squaramide moieties. In various embodiments, the antibody radioisotope construct is Ab-PCTA-Sq-Tb161 or Ab- PCTA-Click-Tb161. In various embodiments, the targeting moiety is specific to the human urokinase plasminogen activator receptor (uPAR). In some embodiments, the antibody radioisotope construct is MNPR- 101-PCTA-Sq-Tb161 or MNPR-101-PCTA-Click-Tb161.

[0098] In some embodiments, the conjugate-to-antibody ratio (CAR) of PCTA to antibody is less than two, such as between 1 and 2, or about 1.5. In some embodiments, embodiments, the antibody radioisotope constructs have a conjugate-to-antibody ratio (CAR) of less than 1 , such as between 0.01 and 1 , or about 0.2. In some embodiments, additional unconjugated (cold) antibody is administered either as part of the diagnostic or therapeutic drug or administered concurrently, before, or after administration of the diagnostic or therapeutic agent.

[0099] Non-limiting examples of the same construct being used for diagnosis and therapy include a therapeutic or sub-therapeutic dose of MNPR-101-PCTA-Sq-Tb-161 sufficient for SPECT imaging and the same or higher dose level for therapy (0- / Auger electron therapy). The use of additional terbium radioisotopes Tb-149 (a-therapy), Tb-152 (PET imaging), and Tb-155 (SPECT imaging) for imaging and therapy can also be used as described by Van Laere (Van Laere et al, Theranostics. 2024; 14(4): 1720-1743). The use of the same radioisotope or a different isotope of the same chemical element is preferable as the chemical structure is identical between imaging and therapy, and the resulting biodistribution of the constructs are also importantly the same.

[0100] Additional non-limiting examples of the same construct include a subtherapeutic or therapeutic dose of MNPR-101-PCTA-Lu-177 for SPECT imaging and the same or higher dose level for therapy. Additional non-limiting examples of the same chemical construct include MNPR-101-PCTA-Cu-64 for PET imaging and MPRP- 101-PCTA-CU-67 for therapy.

[0101] Different chemical constructs employing different bifunctional chelators may also be used for imaging and therapy. Non-limiting examples of different constructs include MNPR-101-DFO*-89Zr as a PET imaging diagnostic agent followed by MNPR-101-PCTA-161Tb for therapy.

[0102] In each of the prior examples, one or more doses of imaging agent may be given prior to, concurrently, or subsequently to one or more doses of the agent.

[0103] Furthermore, a subsequent imaging dose may be administered following a therapeutic dose to assess the efficacy of the therapeutic dose. For example, an imaging dose of MNPR-101-PCTA-89Zr may be administered from 1 week to 12 weeks and preferably 4 to 8 weeks following administration of a therapeutic dose of MNPR-101-PCTA-Ac-225 to assess the efficacy of the therapeutic dose on the disease. In some embodiments, such as MNPR-101-PCTA-Tb-161 for example, a subsequent therapeutic dose can be imaged to assess efficacy of a prior one.Drug Products

[0104] Also provided herein are drug products comprising a radiopharmaceutical or pharmaceutical composition disclosed herein and a non-radioactive molecule specific for the same targeting moiety as the radiopharmaceutical.

[0105] In various embodiments, the drug products further comprise one or more additional agents. In some cases, the one or more additional agents comprise DNA-damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, radiosensitizers, CAR T-cell therapies, chemoagents, radioprotectants, or a combination thereof. In some cases, the radiopharmaceutical or pharmaceutical composition and the one or more additional agents are co-formulated. In some cases, the radiopharmaceutical or pharmaceutical composition and the one or more additional agents are formulated separately. In some cases, the radiopharmaceutical or pharmaceutical composition and the one or more additional agents are packaged together. In some cases, the radiopharmaceutical or pharmaceutical composition and the one or more additional agents are packaged separately.

[0106] In some cases, the drug products comprise one or more radioprotectants. In some cases, the one or more radioprotectants comprise ascorbic acid, gentisic acid, N-acetyl cysteine, human serum albumin, one or more unconjugated “cold” antibodies, or a combination thereof.

[0107] In some cases, the drug products comprise one or more unconjugated “cold” antibodies. Cold antibodies are described in more detail in the section “Cold Antibodies” below. In some cases, the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are the same. In some cases, the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are different.

[0108] In some cases, the drug products further comprise a pharmaceutically acceptable carrier.

[0109] In some cases, the drug products have a defined total antibody mass dose. In some cases, the total antibody mass dose is about 1 mg to about 80 mg. In some cases, the total antibody mass dose is about 2 mg to about 30 mg. In some cases, the total antibody mass dose is about 4.5 mg to about 20 mg. In some cases, the total antibody mass dose is about 10 mg.

[0110] In some cases, the drug products have a defined conjugate to antibody ratio (CAR) with respect to the sample of interest. In some cases, the conjugate to antibody (CAR) ratio is less than 1.Cold Antibodies

[0111] In some embodiments, the antibody radioisotope constructs of the disclosure are provided with a “hot” kit and a “cold” kit. A “cold” kit comprises the mAb conjugated to a chelator that is combined with the “hot” kit, a pharmaceutically acceptable formulation of the radionuclide just prior to administration to a patient for diagnosis or therapy.

[0112] Targeted antigens, such as uPAR, often have low-level expression in normal tissue (Zhai et al. Journal of Translational Medicine (2022) 20:135) which can impact the biodistribution of the administered diagnostic or therapeutic construct, especially those with high target affinity, such as monoclonal antibodies.

[0113] At very low doses of antibody, this normal tissue expression of the target captures a significant portion of the circulating antibody, increasing background uptake, accelerating clearance, and reducing the antibody available to the tumor in an effect known as Target-Mediated Drug Distribution (TMDD) (Ponte et al., Mol Cancer Ther 2021 ;20:203— 12). In a study by Cao et al, the modeled and observed impact of TMDD is shown for several antibodies, and examples of concentration-dependent clearance profile for an antibody are shown (Cao et al, J Pharmacokinet Pharmacodyn. 2014; 41(4): 375-387).

[0114] Importantly, because the antibody mass dose for a radiolabeled antibody for imaging or therapeutic applications can typically be quite small (e.g. < 5mg radiolabeled antibody), antibody radiopharmaceuticals are susceptible to TMDD.

[0115] To overcome the effects of TMDD, unconjugated (“cold”) antibody can be administered to reduce the amount of radiolabeled antibody or peptide captured in normal tissue and allow the radiolabeled antibody or peptide to reach the tumor. Added cold antibody or peptide helps to saturate this low-level expression of the target in normal tissue without saturating the tumor, where target expression is much higher.

[0116] For example, in a study of patients with IGF-1R-expressing advanced solid tumors by Pandit-Taskar (Pandit-Taskar et al., J Nuc Med June 2023;64 suppl :P630 SNMMI 2023 poster presentation), the addition of cold antibody improved image quality via higher tumor-to-background ratio, reduced off-target uptake, and was estimated to increase the therapeutic tumor dose up to 2x while staying within accepted organ safety thresholds.

[0117] Multiple published studies exploring cold antibody dosing with imaging radiopharmaceuticals have similarly demonstrated that cold antibody increases image quality, with the optimal dosing generally falling in the2 - 80 mg (e.g., 20 - 80 mg, 10 - 50 mg, or 20 - 50 mg) range. (Pandit-Taskar N et al, J Nucl Med. 2024 Jul 1 ;65(7):1051 -1056; Morris MJ et al, Clin Cancer Res. 2007 May 1 ;13(9):2707-13.).

[0118] In a preclinical study by Kalidindi et al (Kaladindi et al, Eur J Nucl Med Mol Imaging. 2021 Jul;48(8):2642-2651), the addition of cold peptide was also proven beneficial to the biodistribution of a radiolabeled peptide. Cold peptide can similarly be added to a peptide-based radiopharmaceutical to overcome the effects of TMDD.

[0119] A cold antibody may be used with the antibody radioisotope constructs described herein and drug products comprising the same to improve biodistribution of the antibody radioisotope constructs. In other cases, a cold antibody may play the role of a radioprotectant when administered with the antibody radioisotope constructs described herein and drug products comprising the same.

[0120] Expression data using MNPR-101 staining in a microarray study to assess uPAR expression in normal tissue (Table 4) shows low-level expression of uPAR in normal tissue, indicating that the addition of unconjugated “cold” antibody would benefit the biodistribution of an MNPR-101 radiopharmaceutical.

[0121] Table 4: Summary of uPAR Staining with MNPR-101 in Human Frozen Tissue Microarray

[0122] x / 5 represents the number of the 5 donors whose tissue was positive. The frequency of positive cells is shown as: VR = very rare (1-5%), R = rare (>5%-25%), 0 = occasional >25%-50%), OF = occasional frequent (>50%-75%), and F = frequent (>75%-100%). The staining intensity is represented on a scale of 1 to 4: 1 = weak, 2 = moderate, 3 = strong, and 4 = intense. N / A = not applicable.

[0123] It may be advantageous to include cold antibody into the final drug product formulation for simultaneous dosing of radiolabeled “hot” and unconjugated “cold” antibody to avoid added time and burden to the patient through repeated dose administration. The inclusion of cold antibody in the product vial also serves to protect the radiolabeled antibody from radiation damage, thereby improving the overall stability of the antibody radioisotope construct.

[0124] For example, the final drug product may include the radiolabeled antibody, such as MNPR-101-PCTA- Sq-Tb-161 or other radioisotopes and linkers described herein, 5 mg - 100 mg of cold MNPR-101, a pharmaceutically acceptable carrier or buffer for sterile injection such as sterile saline for example, and optionally at least one or more radioprotectants such as ascorbic acid, gentisic acid, N-acetylcysteine, human serum albumin, one or more unconjugated “cold” antibodies, and others known to those skilled in the art.

[0125] In various embodiments, the amount of unconjugated “cold” antibody, such as MNPR-101, administered may be from about 5 mg to about 100 mg (e.g., from about 5 mg to about 100), about 20 mg to about 40 mg, about 4.5 mg to about 30 mg, or about 10 mg to about 20 mg. The timing of cold (unlabeled) antibody administration may be pre-dosing, post-dosing, or concurrent dosing (including when the cold antibody is formulated in the drug product together with the radiopharmaceutical) of the radiolabeled “hot” antibody, such as MNPR-101 -PCTA-Sq-Ac-225 or MNPR-101-PCTA-Sq-Tb-161.

[0126] The cold antibody may also be included in a kit comprising the final drug product vial or other acceptable container of the formulation for MNPR-101 -PCTA-Sq-Tb-161 or other MNPR-101 compounds or constructs described herein in an appropriately shielded container, a vial or other acceptable container of cold MNPR-101 antibody or other constructs in a sterile, pharmaceutically acceptable carrier for injection such as saline, and optionally, one or more syringes and syringe filters for extraction and injection.

[0127] The amount of cold antibody may be personalized for each patient. With susceptibility to rapid clearance from the blood of the radiopharmaceutical caused by TMDD described above, pharmacokinetic parameters from one or more injections of a targeting agent in a patient may be used to assess whether subsequent doses should maintain, reduce, or increase the total antibody mass dose for that patient. For example, an estimate of the amount of drug in the blood at a given timepoint from a single imaging or therapeutic dose can be assessed to determine whether the level of antibody mass dose should be maintained, increased, or decreased.

[0128] Changes to the antibody mass dose can be accomplished by any of the following means: increasing or reducing the amount of unconjugated “cold” antibody administered; increasing or reducing the specific activity of the radiolabeled antibody; or a combination of the two.

[0129] Using the antibody-based radioisotope constructs described in this disclosure, the construct may be administered, and one or more pharmacokinetic measurements may be performed by measuring or estimating the radioactivity level in blood. Direct measurement of timely collected blood / serum samples can assess the amount of dose (% Injected Dose I ml) in the blood at one or more timepoints. Alternatively, the blood activity can be estimated via another means such as from a volume of the aortic blood on a PET scan or other methods known to those in the art.

[0130] Pharmacokinetic (PK) parameters such as the Area under the curve (AUC), Maximum concentration (Cmax), Time to reach Cmax (Tmax), elimination half-life (t1 / 2), and others can be calculated by assessing radioactivity in the blood and / or serum collected at designated time points after administration of the radiopharmaceutical.

[0131] For example, measurement of 2 time points on the day of drug administration and another at day 3 and a fourth at day 7 after administration would be sufficient to estimate a binomial curve of drug concentration in the blood over time. This curve may be compared to a preferred curve and then the antibody mass dose may be increased or decreased.

[0132] Additional or fewer timepoints may also be assessed. For example, the amount of radiopharmaceutical in the blood at a single timepoint such as an imaging scan between 3 and 10, preferably between 4 and 7 days (e.g., between 5 and 7 days) after injection of an imaging agent (non-limiting examples of which include MNPR- 101-DFO*-Sq-89Zr or MNPR-101-PCTA-Sq-89Zr) may be assessed to determine if a sufficient quantity of drug, such as at least approximately 3% injected dose per liter of plasma (3% I D / L) , remains in circulation at a desired timepoint, for example at 7 days post-injection. If the circulating dose is significantly higher than the target (e.g. if it is 20% or 30% I D / L in the plasma 7 days after injection), the total antibody mass dose for a subsequent administration such as a therapeutic administration (non-limiting examples include MNPR-101-PCTA-Sq-Tb-161 or MNPR-101-PCTA-Sq-Ac-225) would be decreased, and if the circulating dose is lower than the target (e.g. 3% I D / L 4 days after injection), the antibody mass dose would be increased. The target circulating dose is specific to each day and is expected to be higher at earlier measurement time points. For administration of MNPR-101, a total antibody mass dose ranging from 2 mg - 100 mg (e.g., 5 mg - 100 mg, or 5 mg to 80 mg) may be used, which covers the dosing range where TMDD typically has a strong effect.

[0133] Co-treatment: In addition to being a monotherapy, the radiopharmaceutical drugs described herein can also be used in combination therapies to improve efficiency of the radiopharmaceutical or to co-attack the cancer cells (Cornelissen et al. J Nucl Med. 2020 Nov; 61(11): 1544-1552). Therapeutic compositions may comprise, in addition to the antibody radioisotope constructs, one or more additional drugs.

[0134] Examples include but are not limited to radiosensitizers like fluoropyrimidines, gemcitabine, capecitabine, platinum analogs (McGinn et al. Seminars in Radiation Oncology, Volume 13, Issue 1, 2003, Pages 13-21, ISSN 1053-4296), which allows tumor cells to be more sensitive to radiation, potentially improving effectiveness of the radiopharmaceutical and reducing unwanted toxicities. Radiosensitization can be achievedby using drugs that inhibit DNA damage repair, example include but not limited to poly (adenosine diphosphate ribose) polymerase [PARP] inhibitors (Sade et al., Ecancermedicalscience. 2021; 15: ed118) like Veliparib, inhibitors of other DNA damage response (DDR) proteins like DNA-PK inhibitor, example peposertib (Scott et al. JNM June 2023, 64 (supplement 1) P1271). This also includes but not limited to mTOR inhibitors like Rapamycin, Everolimus, AKT inhibitors, PI3K inhibitors, heat shock protein 90 inhibitors, and checkpoint kinase 1 inhibitors which also be used (Beggs et al. Cancers (Basel) 2020 May; 12(5): 1278). Inhibitors of proteins involved in DNA repair like DNA topoisomerases I and II can also induce radiosensitization. In addition to DNA- damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, can be used in combination.

[0135] Other chemotherapeutic agents, CAR T-cell therapies, etc. may also be used to target multiple cancercausing and progressing pathways. These drugs may be co-administered or administered one after the other, or compatible drugs may be assembled in a single or multiple delivery system.

[0136] The radiopharmaceutical may also be paired with external radiotherapy to improve efficacy.

[0137] To improve the distribution of the antibody radioisotope constructs, use of blood flow modulators such as vasodilators like acetazolamide may also be employed.

[0138] In some cases, the methods of treatment disclosed herein further comprise administering to the patient one or more additional agents. In some cases, the one or more additional agents comprise a DNA-damage repair inhibitor. In some cases, the one or more additional agents comprise DNA-damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, radiosensitizers, CAR T-cell therapies, chemotherapeutic agents, radioprotectants, or a combination thereof.

[0139] In some cases, the one or more radioprotectants comprise ascorbic acid, gentisic acid, N-acetyl cysteine, human serum albumin, one or more unconjugated “cold” antibodies, or a combination thereof.

[0140] In some cases, the one or more additional agents comprise one or more unconjugated “cold” antibodies. In some cases, the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are the same. In some cases, the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are different.

[0141] In some cases, administering a therapeutically-effective amount of the radiopharmaceutical or pharmaceutical composition comprises the steps of:(a) administering a mass dose of the radiopharmaceutical or pharmaceutical composition;(b) assessing one or more pharmacokinetic properties of the radiopharmaceutical or pharmaceutical composition; and(c) adjusting a subsequent mass dose of the radiopharmaceutical or pharmaceutical composition based on the assessing in step (b). In some cases, the assessing step (b) comprises(I) assessing one or more pharmacokinetic parameters in the patient after administration of theradiopharmaceutical or pharmaceutical composition; and(ii) determining whether a subsequent dose of the radiopharmaceutical or pharmaceutical composition should maintain, reduce, or increase the total antibody dose for the patient; and the adjusting step (c) comprises maintaining, reducing, or increasing the total antibody dose for the patient based on the determining step (ii).Methods of Diagnosis

[0142] A method of diagnosing includes administering a diagnostically effective amount of a radioisotope construct described herein, e.g., an MNPR-101-PCTA-conjugated, measuring the level of radiation in a first tissue and a second tissue, and diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater, such as 2:1 or greater. The level of radiation emitted by the radioisotope construct can be measured in the patient at one or more times from 10 minutes to 30 days after drug administration using Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET) diagnostic imaging. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater, and the second tissue is liver tissue. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater, and the second tissue is skeletal muscle tissue.

[0143] In some cases, provided are methods of diagnosing a disease or disorder in a patient, comprising the steps of:(a) administering to the patient an effective amount of a radiopharmaceutical or pharmaceutical composition disclosed herein;(b) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 10 minutes to 30 days after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and(c) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater, and the second tissue is liver tissue. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater, and the second tissue is skeletal muscle tissue.

[0144] A method of assessing the aggressiveness of disease includes administering a diagnostically effective amount of an Ab-PCTA-conjugated radioisotope, measuring the level of radiation in a first tissue and a second tissue, and determining the aggressiveness of the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater. In some cases, the ratio of radiation measured in thefirst tissue compared to the second tissue is 1.5:1 or greater, and the second tissue is liver tissue. In some cases, the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater, and the second tissue is skeletal muscle tissue.Kits

[0145] As an additional aspect, the disclosure Includes kits which comprise one or more compounds or compositions packaged In a manner which facilitates their use to practice methods of the disclosure. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising a radiopharmaeutical, pharmaceutical composition, or drug product as described herein), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay. Preferably, the kit contains a label that describes use of the inhibitor compositions. In various embodiments, a kit comprises a radiolabeled antibody as described herein as well as a cold, unlabeled antibody for use in diagnostic or therapeutic methods described herein.

[0146] In a further embodiment, the disclosure provides an article of manufacture, or unit dose form, comprising: (a) a composition of matter comprising a radiopharmaceutical as described herein; (b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said radiopharmaceutical in the treatment or diagnosis of cancer as described herein.

[0147] It is to be understood that while the disclosure is read in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.EXAMPLESGeneral syntheses of PCTA-Sq and PCTA-Click-Sg

[0148] Synthesis of PCTA-Sq can be carried out using methods known to those in the art. Nonlimiting examples of the syntheses of PCTA-Sq are shown in Schemes 1 , 2, and 3.Scheme 1

[0149] Synthesis of PCTA-Click-Sq can be carried out using methods known to those in the art. Nonlimiting examples of the syntheses of PCTA-Click-Sq are shown in Scheme 3, where Ab represents a targeting moiety (e.g., an antibody). In some embodiments, the click complementary pairs can be swapped between PCTA and Squaramate and / or Squaramide.Scheme 3

[0150] A skilled artisan will understand how to select appropriate reagents and conditions for the syntheses in these Schemes.General syntheses of Antibody PCTA-Sg and Antibody PCT A-Click-Sg conjugates

[0151] Synthesis of antibody PCTA-Sq conjugates can be carried out using methods known to those in the art. Nonlimiting examples of the syntheses of PCTA-Sq are described below.

[0152] Synthesis and Characterization of Antibody PCTA-Sq conjugate with Tb-161 (Conjugate B1)-. The preparation of an antibody, such as MNPR-101, with PCTA-Sq may be accomplished in a method similar to the conjugation of DFO*-Sq in Rudd et al., (Chem. Commun. 2016; 52: 11889-11892)and USSN 63 / 570,189).

[0153] Radiolabeling and purification to obtain antibody radionuclide conjugate, e.g., MNPR-101 -PCTA-Sq-161Tb, is performed in the same way as other radioimmunoconjugates (Eur J Nucl Med Mol Imaging. 2014 Oct;41 (10)1907-15, Bioconjugate Chem. 2021 , 32, 7, 1315-1330).

[0154] Synthesis and Characterization of Antibody PCTA-Click-Sq conjugate with Tb-161 (Conjugate B2): In one embodiment, Ab-PCTA-Click-Sq-Tb-161 synthesis (e.g., MNPR-101 -PCTA-Click-Sq-Tb-161) may occur in several steps. PCTA is reacted with a molecule with azide. Another molecule containing a strained octyne group is reacted to add squaramate. The antibody is conjugated with click element containing squaramate (e.g., using the reaction modified from Rudd et al., Chem. Commun. 2016; 52: 11889-11892 and USSN 63 / 570,189). The antibody with squaramate and the alkyne group is then reacted with PCTA containing azide group to perform click binding and form Ab-PCTA-Click-Sq.

[0155] Radiolabeling and purification to obtain MNPR-101-PCTA-Click-161Tb is performed in the same way as for the other radioimmunoconjugates (Eur J Nucl Med Mol Imaging. 2014 Oct;41 (10)1907-15, Bioconjugate Chem. 2021, 32, 7, 1315-1330).Example 1 Potential instability with p-SCN-Bn-PCTA (PCTA-NCS)

[0156] uPAR-targeting antibody MNPR-101 was conjugated to the bifunctional chelator p-SCN-Bz- PCTA ( or p-SCN-Bn-PCTA or PCTA-NCS) and radiolabeled with Tb-161. The phenyl isothiocyanate of PCTA-NCS conjugates to the antibody or antibody fragment via a thiourea bond. Ideally, this thiourea bond forms a stable bond with the amine side chain of a lysine residue on the antibody. However, isothiocyanate can also conjugate to antibodies via the cysteine residue on the antibody, forming a less stable dithiocarbonate (Petri et al. RSC Adv., 2020; 10: 14928-14936) that may be susceptible to cleavage in vivo.

[0157] The selectivity of the conjugation to the preferred lysine is predominately determined by the pH of the reaction solution with higher pH (pH 9.0-11) being desirable while cysteine binding increases at pH 6-8 (Petri et al. RSC Adv., 2020; 10: 14928-14936). However, even at higher pH, some binding to the cysteine still occurs, and variations in the pH during manufacture can lead to unacceptably high levels of weaker cysteine binding. This is undesirable because it has been found with various isothiocyanate derivatives that the cysteine adducts can break apart and bind elsewhere (Karlsson et al.Sci Rep. 2016; 6: 21203), which can cause separation and loss of the conjugated DFO (and its chelated Tb-161) from the antibody.

[0158] This breaking apart and binding elsewhere of isothiocyanate conjugated to cysteine may not be readily detected in in vitro stability assay of conjugated antibody because reattachment can occur to lysines or other amines on the same antibody. However, with respect to in vivo study, the rearrangement of isothiocyanate conjugated to cysteine has far more competing options to reform elsewhere. This will result in rapid breakdown and clearance. This was observed with PCTA-NCS conjugated to the uPAR-targeted antibody, MNPR-101. Similar results were recently observed with a different bifunctional chelator p-SCN-Bn-DFO conjugated to the uPAR-targeted antibody, MNPR-101, included in Provisional Patent Application US 63 / 570,189 for MNPR-101- DFO*-89Zr.

[0159] As an example, in vitro and in vivo Instability of MNPR-101-PCTA-Tb-161 was evaluated using the bifunctional chelator p-SCN-Bn-PCTA (Macrocyclics, Inc). MNPR-101-PCTA-NCS conjugate was chelated to terbium-161 to form MNPR-101-PCTA-NCS-Tb-161. MNPR-101-PCTA-NCS-Tb-161 was synthesized using a 1 :4 stochiometric ratio of MNPR-101 to p-SCN-Bz-PCTA using a process consistent with prior patent on PCTA. Thin layer chromatography (TLC) on the product prior to purification showed >99% labeling at specific activity 10 mCi / mg, 50 mCi / mg and 200 mCi / mg. The synthetic process includes two distinct purification steps by PD-10 column, one following conjugation and a second following chelation to remove unconjugated PCTA, Tb-161- PCTA, and any free Tb-161. Testing of the purified MNPR-101-PCTA-NCS-Tb-161 was performed using radio high performance liquid chromatography (rad-HPLC) after manufacturing and prior to its injection into mice. The results are provided in Table 5. Chromatograms for the UV and Rad signals are collected from the same sample injection. The time and pattern of the chromatogram showed that the antibody was successfully radiolabeled with Tb-161.

[0160] Table 5: HPLC data of MNPR-101 and MNPR-101-PCTA-NCS-161Tb

[0161] TLC after 5 days showed 97% labeling, results provided in Table 6.

[0162] Table 6: Thin layer chromatography (TLC) data of MNPR-101-PCTA-NCS-181Tb

[0163] The data in Table 5 is for MNPR-101-PCTA-NCS-181Tb after labeling. HPLC shows that the MNPR- 101-PCTA-NCS conjugate entities are labelled with Tb-161, with nearly identical peaks shifted slightly later in retention time due to the physical placement of the rad detector following the HPLC’s UV detector. Notably absent were any other significant peaks suggesting nearly all radioisotopes were chelated and attached to MNPR-101.Example 2 In vivo characterization of MNPR-101-PCTA-NCS-Tb-161.

[0164] Following testing, MNPR-101-PCTA-NCS-Tb-161 was then injected into mice (tumor-free) to evaluate its in vivo stability and dosimetry. The whole-body retention In mice was estimated using naive immunocompromised mice administered with approximately 100 pCi of MNPR-101 -PCTA-NCS-161Tb and sacrificed at 2 hours, 1 day, 2 days, 6 days, 9 days, and 21 days post-injection. Whole body retention of the radioisotope was readily measured by placing the entire mouse in a well counter, and mouse excretions were also collected and measured to cross-check whole body retention measurements.

[0165] The mouse whole body retention results of MNPR-101-PCTA-NCS-Tb-161 were then compared with similar experiments in which 210 pCi of MNPR-101-DFO-NCS-89Zr and 100 pCi of MNPR-101-DFO*-Sq-89Zr were administered. Results showed that approximately 47% of the MNRP-101-PCTA-NCS-161Tb was cleared within 24 hours of administration, which is similar to MNPR-101-DFO-NCS-89Zr. MNPR-101-DFO‘-Sq-89Zr, which binds to the antibody using a stable squaramide bond that is specific to lysines and hence is not at risk of weaker cysteine binding, showed less than 10% clearance as disclosed in U.S. Provisional Patent Application No.63 / 570,189). Furthermore, the level of circulating activity of squaramide-bound MNPR-101-DFO*-Sq-89Zr was significantly higher at all timepoints (Figure 1) relative to the isothiocyanate-bound MNPR-101 -PCTA-NCS-Tb- 161 and MNPR-101-DFO-NCS-Zr-89. The rapid loss of drug from the mouse in the first 24 hours suggests that the portion of the bi-functional chelator bound to the cysteine rapidly separates in vivo and is excreted, while the stable isothiocyanate-lysine bound radiopharmaceutical remains in the body for a significantly longer time period.

[0166] Results from MNPR-101-DFO*-Sq-89Zr showed a significant improvement in tumor uptake. Similar increased bioavailability may be achieved using MNPR-101-PCTA-Sq-161Tb instead of MNPR-101-PCTA-NCS-161Tb.

[0167] This breaking apart and binding elsewhere of isothiocyanate conjugated to cysteine may not be detected in in vitro stability assays of conjugated antibody (as shown in Fig 1 and 2) because reattachment can occur to other cysteines, lysines or other amines on the same antibody. However, with respect to in vivo studies, the rearrangement of isothiocyanate conjugated to cysteine has far more competing options to reform elsewhere and results in rapid breakdown and clearance.Example 3 Antibody radioisotope constructs using PCTA-Sa and PCTA-Click for MNPR-101-PCTA-Sa-Tb161

[0168] In prior radiopharmaceutical studies with Terbium-161, the chelator DOTA (2,2',2",2"'-(1 ,4,7,10- Tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) has been and continues to be among the most commonly-used chelators in preclinical studies.(Eur J Nucl Med Mol Imaging. 2014 Oct;41 (10):1907-15, Front Med (Lausanne). 2021; 8: 675122.)

[0169] As disclosed above, the radioisotope Tb-161 chelated with the bi-functional chelator p-SCN-Bn-PCTA bound to MNPR-101 was used to form MNPR-101-PCTA-SCN-Tb-161 and assessed its performance in vivo. Schematics of the antibody conjugates are set out in Table B. No prior chelation of any radioactive isotopes of Terbium to PCTA or the in vivo assessment of PCTA-Tb-161 conjugated to a targeting agent is known to date. Figure 2 shows SPECT imaging of MNPR-101-PCTA-NCS-Tb-161 administration to immunocompromised mice implanted with uPAR expressing pancreatic cancer cells, MIA PaCa-2. Although a portion of the Tb-161 in the form PCTA-Tb-161 was rapidly cleared via the bladder as seen at the 2h timepoint in Fig 4 due to the weak cysteine binding of the NCS isothiocyanate bond discussed above, the uPAR-positive tumor showed robust and durable uptake of the remaining MNPR-101-PCTA-NCS-Tb-161 through at least nine days with limited off-target organ uptake. Notably absent is any significant decrease of Tb-161 in the tumor or accumulation of free Terbium- 161 in the liver, the organ where other lanthanides (e.g., free Lu-177) are known to accumulate. Both observations suggest excellent in vivo stability of the PCTA-Tb-161 chelation.Example 4: Low PCTA Conjugate-to-Antibodv Ratio (CAR) of 5 or less

[0170] In tumor-bearing immunocompromised mice, in vivo biodistribution of different PCTA chelator-to- antibody ratios (CAR) using p-SCN-Bn-PCTA (Macrocyclics Plano, Tx) was evaluated. To do so, I ndium-111 was chelated using PCTA to an lgG1 monoclonal antibody, MNPR-101, which targets with high affinity the urokinase plasminogen activator receptor (uPAR) expressed in multiple aggressive cancers.

[0171] Xenograft tumors were grown in mice using the triple negative breast cancer cell line, MDA-MB-231 , which expresses uPAR. The radioisotope component of MNPR-101-PCTA-ln111 (In 111) enables single-photon emission computed tomography (SPECT) imaging of the tumors and ex-vivo biodistribution after sacrifice using gamma detectors.

[0172] With comparable total injected antibody mass and similar specific activities, a radiolabeled antibody with a low chelator-to-antibody ratio < 2:1 (“Low CAR PCTA”) as well as a radiolabeled antibody with a higher chelator-to-antibody ratio of approximately 8:1 (“High CAR PCTA”) have been produced. Binding affinity kinetics measurements of MNPR-101 compared to MNPR-101-PCTA conjugates with suPAR by Surface Plasmon Resonance studies, showed that the conjugates had comparable KD value compared to MNPR-101. However, a comparison of these in vivo produced dramatically larger and unexpected differences in biodistribution.Surprisingly and unexpectedly, the Low CAR PCTA produced significant improvements in desired biodistribution with significantly improved tumor uptake (was off target, e.g., liver comparable to 12:1 or better too), relative to a more typical High CAR PCTA. Importantly, because MNPR-101 is specific only to human uPAR, which is expressed only the human xenograft tumor from the MDA-MB-231 cell line, and does not bind to murine uPAR, the changes in the relative biodistribution can be attributed to changes in the binding ability of the antibody to the target as well as its propensity for non-specific (off-target) binding.

[0173] A comparison of biodistribution is shown in Figure 3.Example 5 Evaluation of Coniuqate-to-Antibodv Ratio on Immuno-reactivitv In Vitro

[0174] MNPR-101-PCTA conjugated at various combination ratios were evaluated for binding affinity kinetics with uPAR to identify if higher CAR affects naked antibody affinity to uPAR. This effect of CAR on affinity may be consequential in vivo, affecting tumor uptake and general biodistribution in both diagnostic and therapeutic applications.

[0175] Table 7: Surface Plasmon Resonance (SPR) evaluation of MNPR-101 and MNPR-101-PCTA conjugates binding affinity kinetics with suPAR.

[0176] Binding kinetics of MNPR-101 and MNPR-101-PCTA conjugates and its antigen, suPAR was evaluated by SPR using GE Biacore platform. KD is inversely proportional to affinity. MNPR-101 and PCTA were conjugated at various molar ratios. Conjugate-to-Antibody Ratios (CAR) of up to 5:1 show less than a factor of 2 reduction in binding affinity while higher ratios show more significant declines in binding affinity.Example 6: Synthesis and Characterization of MNPR-101-PCTA-NCS-Ac-225 at different CAR requires a lower specific activity

[0177] In the work of Mazar et al. (U.S. Patent Application Nos. 17 / 749,574 and 17 / 749,763), MNPR-101- PCTA-NCS-Ac225 preparation used a specific activity of 1 mCi / mg and a CAR of 12:1. However, based on the binding affinity data above and the MNPR-101-PCTA-ln-111 data also shown above, a more desirable CAR of 5:1 or less is preferable for Ac-225. The below data with PCTA-Ac-225 shows a lower CAR with much lower labeling efficiency. In labeling experiments, the antibody radioisotope construct MNPR-101-PCTA-Ac-225 using a 2:1 CAR could not achieve 1 mCi / mg. Advantageously, the present example required a specific activity of 0.1 mCi / mg for labeling, which labeled less efficiently even at a 10x lower activity than the 1 mCi / mg used by Mazar for the MNPR-101-PCTA with a CAR of 12:1.

[0178] MNPR-101-PCTA conjugates were labelled with Ac-225 at an estimated specific activity of 0.1 mCi / mg.

[0179] Table 8: Thin layer chromatography (TLC) data of MNPR-IOI-PCTA-NCS-^AcExample 7: Synthesis of PCTA-Sq

[0180] Synthesis of PCTA-Sq was carried out using the reaction scheme shown above. 3.5 mg of PCTA-NH2 • 2 HCI • 2 H2O (5.9 mmol) was dissolved in 1 mL of Ethanol and 1.0 mg diethyl squarate (5.9 mmol) and 1.0 mg of DIPEA was added to the solution. The reaction was stirred at room temperature for 2 hours.

[0181] HPLC analysis on the reaction product revealed one major product and MS analysis of this identified the presence of the expected product (PCTA-Sq) with appropriate molecular weight((M+H)+= 610).Example 8: Effect of Antibody PCTA-Sq conjugates labeled with Lu-177 in a colon cancer model

[0182] Tumor-bearing immunocompromised mice were dosed with Trastuzumab-PCTA-Sq-177Lu, Sacituzumab-PCTA-Sq-177Lu or Labetuzumab-PCTA-Sq-177Lu, and the tumor uptake of the conjugates were evaluated using imaging methods.

[0183] A colon cancer cell line, HT-29, known to express uPAR as reported in Boonstra M. C., et al., Oncotarget. 2015; 6: 14260-14273, was used to develop a xenograft mouse model in immunocompromised mice. From 80 - 85% confluent culture, showing 90% viability, 3 x 106cells were subcutaneously injected into the lower right flank of 406 week old athymic nude mice.

[0184] Mice with palpable tumors were injected intravenously (iv) with 350 pCi of Trastuzumab-PCTA-Sq-177Lu, Sacituzumab-PCTA-Sq-177Lu or Labetuzumab-PCTA-Sq-177Lu. SPECT images were acquired. The results (FIG. 4) showed tumor uptake of these drugs.Example 9: Synthesis and Characterization of Antibody PCTA-Sq conjugates and labeling with Ac-225

[0185] MNPR-101-PCTA-Sq-Ac225 was synthesized using a 1:2 stochiometric ratio of MNPR-101 to PCTA- Sq using a process modified from Rudd et al., Chem. Commun. 2016; 52: 11889-11892 and US provisional Application No. 63 / 570,189. The process included purification steps by PD-10 column following both conjugation and chelation. For radiolabeling, Ac-225 was added to ammonium acetate buffer at pH 5.8 and then reacted with MNPR-101 -PCTA-Sq for 1.5 hours at 37°C. The specific activity of this reaction is approximately 0.05 mCi / mg, where approximately 400 pCi of Ac-225 was reacted with approximately 8 mg of conjugate with a near 100% yield. Radiochemical yield / purity (RCY / RCP) were assessed after the reaction and post-purification by means of TLC, which resulted in >99% RCY and RCP.

[0186] A pancreatic cancer cell line, MIA PaCa-2, known to express uPAR as reported in Gorantla et al., Mol Cancer Res 2011; 9(4): 377-89, was used to develop a xenograft mouse model in immunocompromised mice. Mice were treated with 250 nCi of MNPR-101 -PCTA-Sq-^Ac and tumor volumes were measured twice weekly for 45 days after injection. Treatment of a pancreatic cancer MIA PaCa-2 xenograft mouse model with 101- PCTA-Sq-Ac225 led to significant tumor shrinkage up to 45 days after a single dose (FIG. 5)

[0187] Further aspects of the disclosure are described in the following numbered paragraphs.Embodiments of the disclosure

[0188] Embodiment 1. A composition of matter of a bifunctional chelator comprising: a) The chelator PCTA; b) a linker comprising a benzene ring and a terminal squaramate; c) and optionally a carbon chain.

[0189] Embodiment 2. A composition of matter of a bifunctional chelator comprising: a) The chelator PCTA;b) a linker comprising a click moiety and a terminal squaramate; c) and optionally a carbon chain.

[0190] Embodiment s. A radiopharmaceutical comprising: a) A targeting moiety; b) A bi-functional chelator comprising: i. The chelator PCTA; ii. a linker comprising a terminal squaramate for binding to the targeting moiety and at least one of a benzene ring and / or click reactive group; and c) A radioactive isotope.

[0191] Embodiment 4. The radiopharmaceutical of Embodiment 3 wherein the targeting moiety is an antibody.

[0192] Embodiment 4a. The radiopharmaceutical of Embodiment 4, wherein that antibody targets uPAR.

[0193] Embodiment 4b. The radiopharmaceutical of Embodiment 4 or 4a, wherein the uPAR-targeting antibody is MNPR-101.

[0194] Embodiment 5. The radiopharmaceutical of Embodiment 3 or 4, wherein the radioactive isotope is Lu- 177, Tb-161, Ac-225, Zr-89, or Sn-117m.

[0195] Embodiment 6. The radiopharmaceutical of Embodiment 3 or 4, wherein the chelator to antibody ratio is <5.

[0196] Embodiment 7. A radiopharmaceutical composition of matter comprising:

[0197] An antibody conjugate as shown in Table C:Table Cwhere M can be Lu-177, Tb-161, Zr-89, Ac-225, orSn-117m.

[0198] Embodiment 8. A method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a radiopharmaceutical construct comprising: a) a targeting moiety specific for human urokinase plasminogen activator receptor (uPAR); b) a radioisotope; and c) a bifunctional chelator wherein the bifunctional chelator comprises a chelator with one or more squaramide moieties.

[0199] Embodiment 8a. The method of Embodiment 8, wherein the targeting moiety may be non-uPAR targeting, antibody, antibody fragment, peptide, ScFv, small molecules, aptamer, etc.

[0200] Embodiment 8b. The method of Embodiment 8 or 8a, wherein the bifunctional chelator contains a click reactive group.

[0201] Embodiment 8c. The method of Embodiment 8, 8a, or 8b, wherein the chelator is one of PCTA, diethylenetriamine pentaacetate (DTPA), deferoxamine (DFO), DFO*, dodecane tetraacetic acid (DOTA), or Macropa.

[0202] Embodiment 8d. The method of Embodiment 8, 8a, 8b, or 8c, wherein a DNA-damage repair inhibitor is administered prior to, concurrent with, or subsequent to administration of the radiopharmaceutical.

[0203] Embodiment 9. A pharmaceutically suitable radiopharmaceutical for injection comprising: Drug product comprising radiopharmaceutical of any one of Embodiments 1-7, unconjugated antibody, and one or more radioprotectants (e.g. gentisic acid, ascorbic acid, N-acetylcysteine, human serum albumin, or one or more unconjugated “cold” antibodies,).

[0204] Embodiment 10. A method of treatment comprising administering a mass dose of radiolabeled targeting agent, assessing at least one pharmacokinetic property of the targeting agent in humans, and adjusting a subsequent mass dose of the targeting agent based on that estimate.

[0205] Embodiment 11. A composition of matter of a bifunctional click-squaramide group (“Click-Sq Handle”) comprising: a) A terminal benzene-squaramate group for conjugation to a targeting agent; b) One or more unreacted click functionalities; and c) A chemical linker connecting the benzene-squaramate to the one or more click functionalities.

[0206] Embodiment 12. A composition of matter of a bifunctional chelator (“B ifu nctional+CI ick Handle”) comprising: a) A terminal benzene-squaramate group for conjugation to a targeting agent; b) One or more unreacted click functionalities;c) A group that can chelate or chemically bond to radioisotopes; and d) A chemical linker connecting the benzene-squaramate, one or more click functionalities, and a group that can chelate or chemically bond to radioisotopes.

[0207] Embodiment 13. A composition of matter of a radiopharmaceutical comprising: a) A targeting agent; b) A Bifunctional+Click Handle as described in Embodiment 12; and c) A radioisotope.

[0208] Embodiment 13a. The composition of Embodiment 13, wherein the targeting agent is an antibody.

[0209] Embodiment 13b. The composition of Embodiment 13 or 13a, wherein the targeting agent is MNPR-101.

[0210] Embodiment 13c. The composition of Embodiment 13, 13a, or 13b, wherein the radioisotope isLu-177, Tb-161, Ac-225, Zr-89, or Sn-117m.

Claims

What is claimed is:

1. A radiopharmaceutical comprising: a targeting moiety; a chelating linker comprising a chelator and a linker moiety; and a radioactive isotope, wherein the linker moiety comprises a terminal squaramate moiety and a CMO aryl ring, a click reactive group, or both.

2. The radiopharmaceutical of claim 1 , wherein the chelator is selected from DOTA or a derivative thereof, NOTA or a derivative thereof, HOPO or a derivative thereof, HEHA or a derivative thereof, macropa or a derivative thereof, TETA or a derivative thereof, and SarAr or a derivative thereof.

3. A radiopharmaceutical comprising: a targeting moiety; a chelating linker comprising 3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11 ,13-triene-3,6,9-acetic acid (PCTA) and a linker moiety; and a radioactive isotope, wherein the linker moiety comprises a terminal squaramate moiety and a CMO aryl ring, a click reactive group, or both.

4. The radiopharmaceutical of any one of claims 1 to 3, wherein the targeting moiety comprises an antibody, antibody fragment, peptide, single-chain variable fragment (scFv), small molecule, or aptamer.

5. The radiopharmaceutical of any one of claims 1 to 4, wherein the targeting moiety comprises an antibody.

6. The radiopharmaceutical of claim 5, wherein the antibody targets human urokinase plasminogen activator receptor (uPAR).

7. The radiopharmaceutical of claim 5 or 6, wherein the antibody is MNPR-101 or MNPR-102.

8. The radiopharmaceutical of claim 7, wherein the antibody is MNPR-101.

9. The radiopharmaceutical of any one of claims 1 to 8, wherein the linker moiety comprises an alkyl spacer, a PEG spacer, or both.

10. The radiopharmaceutical of any one of claims 1 to 9, wherein the linker moiety comprises a C&. 10 aryl ring.

11. The radiopharmaceutical of claim 10, wherein the linker moiety comprises a phenyl ring.

12. The radiopharmaceutical of any one of claims 1 to 9, wherein the linker moiety comprises one or more click reactive groups.

13. The radiopharmaceutical of claim 12, wherein the linker comprises one click reactive group.

14. The radiopharmaceutical of claim 12, wherein the linker comprises two click reactive groups.

15. The radiopharmaceutical of claim 12, wherein the linker comprises three or more click reactive groups.

16. The radiopharmaceutical of any one of claims 12 to 15, wherein the one or more click reactive groups comprise an alkyne moiety.

17. The radiopharmaceutical of any one of claims 12 to 16, wherein the one or more click reactive groups comprise a cyclooctyne moiety or a dibenzocyclooctyne-amine (DBCO-amine) moiety.

18. The radiopharmaceutical of any one of claims 12 to 17, wherein the one or more click reactive groups comprise an azide moiety.

19. The radiopharmaceutical of any one of claims 12 to 18, wherein the one or more click reactive groups comprise a tetrazine moiety.

20. The radiopharmaceutical of claim 19, wherein the one or more click reactive groups comprise a 1 ,2,4,5-tetrazine moiety.

21. The radiopharmaceutical of any one of claims 1 to 20, wherein the radioactive isotope is177Lu,161Tb,225Ac,89Zr,111ln, or117mSn.

22. The radiopharmaceutical of claim 21 , wherein the radioactive isotope is161Tb.

23. The radiopharmaceutical of claim 21 , wherein the radioactive isotope is ^Ac.

24. The radiopharmaceutical of claim 21 , wherein the radioactive isotope is177Lu.

25. The radiopharmaceutical of claim 21 , wherein the radioactive isotope is117mSn.

26. The radiopharmaceutical of any one of claims 1 to 25, having a conjugate-to-antibody ratio (CAR) between 1 and 5.

27. The radiopharmaceutical of claim 26, wherein the conjugate-to-antibody ratio (CAR) is between 1 and 2.

28. The radiopharmaceutical of claim 26 or 27, wherein the conjugate-to-antibody ratio (CAR) is about 1.5.

29. The radiopharmaceutical of any one of claims 1 to 25, having a conjugate-to-antibody ratio (CAR) between 0.01 and 1.

30. A radiopharmaceutical having a structure shown in Table B.

31. An antibody conjugate having a structure shown in Table C.

32. A pharmaceutical composition comprising the radiopharmaceutical of any one of claims 1 to 31 and a pharmaceutically acceptable excipient or carrier.

33. A drug product comprising the radiopharmaceutical of any one of claims 1 to 30, the antibody conjugate of claim 31, or the pharmaceutical composition of claim 32 and a non-radioactive molecule specific for the same targeting moiety as the radiopharmaceutical.

34. The drug product of claim 33, further comprising one or more additional agents.

35. The drug product of claim 34, wherein the one or more additional agents comprise DNA- damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, radiosensitizers, CAR T-cell therapies, chemotherapeutic agents, radioprotectants, or a combination thereof.

36. The drug product of claim 35, further comprising one or more radioprotectants.

37. The drug product of claim 36, wherein the one or more radioprotectants comprise ascorbic acid, gentisic acid, N-acetyl cysteine, human serum albumin, one or more unconjugated “cold” antibodies, or a combination thereof.

38. The drug product of any one of claims 33 to 37, comprising one or more unconjugated “cold” antibodies.

39. The drug product of claim 38, wherein the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are the same.

40. The drug product of claim 38, wherein the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are different.

41. The drug product of any one of claims 33 to 40, further comprising a pharmaceutically acceptable carrier.

42. The drug product of any one of claims 33 to 41 , wherein the total antibody mass dose is about 1 mg to about 80 mg.

43. The drug product of any one of claims 33 to 42, wherein the total antibody mass dose is about 2 mg to about 30 mg.

44. The drug product of any one of claims 33 to 43, wherein the total antibody mass dose is about 4.5 mg to about 20 mg.

45. The drug product of any one of claims 33 to 44, wherein the total antibody mass dose is about 10 mg.

46. The drug product of any one of claims 33 to 45, wherein the conjugate to antibody ratio (CAR) is less than 1.

47. The drug product of claim 46, wherein the conjugate-to-antibody ratio (CAR) is between 0.05 and 1.

48. A method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a thera peutica lly-effective amount of the radiopharmaceutical of any one of claims 1 to 30, the antibody conjugate of claim 31, the pharmaceutical composition of claim 32, or the drug product of any one of claims 33 to 47.

49. The method of claim 48, further comprising administering to the patient one or more additional agents.

50. The method of claim 49, wherein the one or more additional agents comprise a DNA-damage repair inhibitor.

51. The method of claim 49 or 50, comprising administering the one or more additional agents to the patient prior to, concurrently with, or subsequently to administering the radiopharmaceutical or pharmaceutical composition.

52. The method of any one of claims 48 to 51 , wherein the one or more additional agents comprise DNA-damage repair inhibitors, immune checkpoint inhibitors, growth factors, immune system modulators, radiosensitizers, CAR T-cell therapies, chemotherapeutic agents, radioprotectants, or a combination thereof.

53. The method of claim 52, wherein the one or more radioprotectants comprise ascorbic acid, gentisic acid, N-acetyl cysteine, human serum albumin, one or more unconjugated “cold” antibodies, or a combination thereof.

54. The method of claim 52 or 53, wherein the one or more additional agents comprise one or more unconjugated “cold” antibodies.

55. The method of claim 54, wherein the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are the same.

56. The method of claim 54, wherein the one or more unconjugated “cold” antibodies and the antibody of the antibody radioisotope construct are different.

57. The method of any one of claims 48 to 56, wherein administering a therapeutically-effective amount of the radiopharmaceutical or pharmaceutical composition comprises the steps of:(a) administering a mass dose of the radiopharmaceutical or pharmaceutical composition;(b) assessing one or more pharmacokinetic properties of the radiopharmaceutical or pharmaceutical composition; and(c) adjusting a subsequent mass dose of the radiopharmaceutical or pharmaceutical composition based on the assessing in step (b).

58. The method of claim 57, wherein the assessing step (b) comprises(i) assessing one or more pharmacokinetic parameters in the patient after administration of the radiopharmaceutical or pharmaceutical composition; and(ii) determining whether a subsequent dose of the radiopharmaceutical or pharmaceutical composition should maintain, reduce, or increase the total antibody dose for the patient; and the adjusting step (c) comprises maintaining, reducing, or increasing the total antibody dose for the patient based on the determining step (ii).

59. A method of treating a disease or disorder in a patient, comprising the steps of:(a) diagnosing the disease or disorder in the patient, comprising i) administering to the patient an effective amount of the radiopharmaceutical of any one of claims 1 to 30, the antibody conjugate of claim 31 , the pharmaceutical composition of claim 32, or the drug product of any one of claims 33 to 47;(ii) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 10 minutes to 30 days after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and(Hi) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater; and(b) treating the disease or disorder by administering to the patient a therapeutically-effective amount of the radiopharmaceutical of any one of claims 1 to 30, the antibody conjugate of claim 31 , the pharmaceutical composition of claim 32, or the drug product of any one of claims 33 to 47.

60. The method of claim 59, wherein the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater.

61. The method of any one of claims 48 to 60, wherein the disease or disorder is cancer.

62. The method of claim 61, wherein the cancer is one or more of lung cancer, ovarian cancer, breast cancer, gastric cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, pancreatic cancer, or colorectal cancer.

63. A method of diagnosing a disease or disorder in a patient, comprising the steps of:(a) administering to the patient an effective amount of the radiopharmaceutical of any one of claims 1 to 30, the antibody conjugate of claim 31 , the pharmaceutical composition of claim 32, or the drug product of any one of claims 33 to 47;(b) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 10 minutes to 30 days after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and(c) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 1.5:1 or greater.

64. The method of claim 63, wherein the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater.

65. A bifunctional chelator compound comprising a chelator moiety and a linker moiety, wherein the linker moiety comprises a terminal squaramate moiety and a Cs-io aryl ring, a click reactive group, or both.

66. The bifunctional chelator compound of claim 65, wherein the chelator moiety is a cyclic chelator moiety.

67. The bifunctional chelator compound of claim 66, wherein the cyclic chelator moiety comprises DOTA or derivatives thereof, NOTA or derivatives thereof, HEHA or derivatives thereof, macropa or derivatives thereof, TETA or derivatives thereof, or SarAr or derivatives thereof.

68. The bifunctional chelator compound of claim 66 or 67, wherein the cyclic chelator moiety comprises DO2A, DO3A, DOTA-3py, DOTAGA, NODAGA, NOTP, H2BZmacropa, TETPA, DiAmSar, SarAr- NCS, AmBaSar, or BaBaSar.

69. A bifunctional chelator compound comprising 3, 6, 9,15-tetraazabicyclo-[9.3.1 jpentadeca- 1 (15), 11 ,13-triene-3,6,9-acetic acid (PCTA) and a linker moiety, wherein the linker moiety comprises a terminal squaramate moiety and a Ce-io aryl ring, a click reactive group, or both.

70. The bifunctional chelator compound of claim 69, having the structure of Formula (I):wherein each X1is independently a chelating group;RNis H or C1-6 alkyl;R° is C1.6 alkyl; andL is a linker moiety comprising a C6-10aryl ring, a click reactive group, or both.

71. The bifunctional chelator compound of claim 70, having the structure of Formula (la), (lb), or72. The bifunctional chelator compound of claim 70 or 71, wherein each X1is independently COOH, CONH2, PO(OH)2, or a picolinic acid moiety.

73. The bifunctional chelator compound of any one of claims 65 to 67, wherein each X1is independently COOH or PO(OH)2.

74. The bifunctional chelator compound of any one of claims 65 to 68, wherein at least one X1is COOH.

75. The bifunctional chelator compound of any one of claims 70 to 74, wherein each X1is COOH.

76. The bifunctional chelator compound of any one of claims 70 to 75, wherein RNis H.

77. The bifunctional chelator compound of any one of claims 70 to 76, wherein R° is ethyl.

78. The bifunctional chelator compound of any one of claims 70 to 77, wherein the linker moiety comprises an alkyl spacer, a PEG spacer, or both.

79. The bifunctional chelator compound of any one of claims 70 to 78, wherein the linker moiety comprises a C6-10aryl ring.

80. The bifunctional chelator compound of any one of claims 70 to 79, wherein the linker moiety comprises a phenyl ring.

81. The bifunctional chelator compound of any one of claims 70 to 80, wherein the linker moiety comprises one or more click reactive groups.

82. The bifunctional chelator compound of claim 81, wherein the linker comprises one click reactive group.

83. The bifunctional chelator compound of claim 81 , wherein the linker comprises two click reactive groups.

84. The bifunctional chelator compound of claim 81 , wherein the linker comprises three or more click reactive groups.

85. The bifunctional chelator compound of any one of claims 81 to 84, wherein the one or more click reactive groups comprise an alkyne moiety.

86. The bifunctional chelator compound of any one of claims 81 to 85, wherein the one or more click reactive groups comprise a cyclooctyne moiety or a dibenzocyclooctyne-amine (DBCO-amine) moiety.

87. The bifunctional chelator compound of any one of claims 81 to 86, wherein the one or more click reactive groups comprise an azide moiety.

88. The bifunctional chelator compound of any one of claims 81 to 87, wherein the one or more click reactive groups comprise a tetrazine moiety.

89. The bifunctional chelator compound of claim 88, wherein the one or more click reactive groups comprise a 1 ,2,4,5-tetrazine moiety.

90. A bifunctional chelator compound having a structure shown in Table A.