Conjugates for EGFR targeted radiotheranostics

Abstract

The present disclosure provides, inter alia, a conjugate that can be radiolabeled and administered to a subject for diagnosing and treating a cancer associated with overexpression of EGFR, such as, e.g., head and neck squamous cell carcinoma (HNSCC).

Description

Attorney Docket No.: 3014529.000043CONJUGATES FOR EGFR TARGETED RADIOTHERANOSTICSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 691 ,294, filed on September 5, 2024, the entire content of which is hereby incorporated by reference.FIELD OF DISCLOSURE

[0002] The present disclosure relates to, inter alia, a conjugate for radiotheranostics and methods for diagnosing and treating a cancer, such as a cancer associated with EGFR overexpression, in a subject in need thereof using the same.BACKGROUND

[0003] Targeted radionuclide therapy (TRT) is a widely used cancer treatment option that employs radiopharmaceuticals to target and deliver ionizing radiation to kill cancer cells (1 -3). TRTs have been used for cancer therapy demonstrating increased overall survival as exemplified in patients with thyroid cancer, prostate cancer, and neuroendocrine tumours (4). As a frontline type of current cancer therapy, TRT delivers a therapeutic dose of radiation to cancer cells using radioactive drugs (radiopharmaceuticals) labelled with radionuclides such as alpha(a)- or beta(|3')-emitting radioisotopes (3,5). TRT with a emitters (a-TRT) offers several advantages compared to P- emitters, mainly due to the delivery of high-energy a-particles (5-9 MeV) to the tumour with a short pathlength (50-100 pm) and high linear energy transfer (LET), causing less toxicity to neighbouring healthy tissues (6). However, radiopharmaceuticals for a-TRT cannot directly be used for imaging applications in vivo to assess their biodistribution and target binding and retention profile as crucial criteria for patient selection and dose calculation in the clinical setting (1 ). The development and application of radiopharmaceuticals combining targeted imaging and therapy, also called radiotheranostics, represent a rapidly evolving field in oncologic nuclear medicine (8,9). Ideal radiotheranostics use different radioisotopes for imaging and therapy of the same chemical element to ensure similar pharmacokinetics, metabolism andbiodistribution patterns (1 ,8). Typical examples of ideal radionuclide pairs in radiotheranostics include64Cu / 67Cu, ^Y / ^Y,124l / 131l,152Tb / 161Tb,133La / 135La and203Pb / 212Pb (11-14). In addition, physical half-life, availability, and production costs also require special consideration in the design and development of radiotheranostics (10).

[0004] The203Pb / 212Pb radionuclide pair has recently gained much attention for developing radiotheranostics for TRT (12,13).203Pb emits y-photons through electron capture, allowing detection with single-photon emission computed tomography (SPECT) for diagnostic imaging, whereas212Pb decays by emitting |3'-particles and a-particles suitable for delivering therapeutic doses of radiation to cancer cells (13,14).

[0005] Accordingly, there is a need for developing radiotheranostics compounds, compositions, and methods for a-TRT that targets various cancers. Aspects of this disclosure are directed to meeting these and other needs.SUMMARY

[0006] Aspects of the present disclosure relate to a conjugate having the formula A-(L-C)n, wherein A is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR), L is a bond or a linker, C is a lead specific chelating moiety according to Formula 5a:Formula 5a each Ri is independently hydrogen or an alkyl group,R2 is -OR1, -NH2 or a bond connecting the lead specific chelating moiety,C, to the linker, L,R4 and Rs are independently hydrogen or a bond connecting the lead specific chelating moiety to the linker, L, provided (i) at least one of R4 and Rs is hydrogen, and (ii) each of R4 and Rs is hydrogen when R2 is a bond connecting the lead specific chelating moiety, C, to the linker, L, and n is at least 1 .

[0007] In one embodiment, in a case where the other OR1 groups in Formula 5a are each OH, R2 is other than an OH group, such as -NH2, -OR1 where R1 is an alkyl group, or a bond connecting the lead specific chelating moiety, C, to the linker, L.

[0008] Aspects of the present disclosure relate to a process for the preparation of a compound suitable for radiolabeling comprising reacting a binding moiety A that is selective for epidermal growth factor receptor (EGFR), and that is any of a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein, and comprises one or more lysine residues, with a lead specific chelating moiety C corresponding to any of Formulas 3a, 3b and 3c:Formula 3bFormula 3c whereinRi is hydrogen or an alkyl group.

[0009] Aspects of the present disclosure relate to a method of radiolabeling a conjugate corresponding to any of Formula 1a, Formula 1 b or Formula 1 c:Formula 1c whereinA is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR),L is a bond or linker,R3 is -OR1 or -NH2, wherein R1 is an alkyl group, and n is at least 1 , the method comprising: combining the conjugate with a lead radioisotope in an aqueous solution at a temperature of no more than 60°C.

[0010] Aspects of the present disclosure relate to a method of imaging or diagnosing a subject suffering from a cancer associated with overexpression of epidermal growth factor receptor (EGFR), the method comprising administering a conjugate as described herein, as radiolabeled with203Pb (Pb-203), to the subject, and imaging tissue having the radiolabeled conjugate compound bound thereto.

[0011] Aspects of the present disclosure relate to a method of treating a subject suffering from a cancer or other diseases associated with overexpression epidermal growth factor receptor (EGFR), the method comprising administering a conjugate as described herein, as radiolabeled with212Pb (Pb-212), to the subject, in a dosage sufficient to kill tumor cells or diseased cells or tissue.

[0012] A method of diagnosing and treating a subject suffering from a cancer associated with overexpression of EGFR, the method comprising: diagnosing the subject by administering conjugate described herein, as radiolabeled with203Pb (Pb- 203), and imaging tissue having the radiolabeled conjugate compound bound thereto to diagnose the subject as being afflicted with the cancer; and treating the subject diagnosed as being afflicted by the cancer, by administering a conjugate as described herein, as radiolabeled with212Pb (Pb-212), in a dosage sufficient to kill cancer cells.

[0013] Aspects of the present disclosure relate to the use of the conjugates as described herein, as radiolabeled with203Pb (Pb-203), to the subject for medical imaging and / or diagnosis or monitoring of a subject suffering from cancer.

[0014] Aspects of the present disclosure relate to the use of the conjugates as described herein, as radiolabeled with212Pb (Pb-212), for treatment of a subject suffering from cancer.

[0015] Aspects of the present disclosure relate to an aqueous formulation comprising a conjugate as described herein, as radiolabeled with a lead radioisotope, wherein the formulation comprises a buffer and optionally further comprises a stabilizer comprising any one or more of ascorbic acid, sodium acetate, gentisic acid, a chelator (e.g. DTPA or DSMA), and ethanol.

[0016] Aspects of the present disclosure relate to the development and evaluation of203Pb-labelled panitumumab (203Pb-PSC-panitumumab) as an immuno- SPECT radioligand for the detection of EGFR+ cancers such as, e.g., head and neck squamous cell carcinoma (HNSCC) in a subject in need thereof. The 51.9 hours physical half-life and favourable y-emission (279 keV; 81 %) of203Pb offer an excellent opportunity for developing immuno-SPECT radioligands. Moreover,203Pb has a complementary therapeutic radionuclide (212Pb), making203Pb and212Pb an ideal matched radiotheranostic pair.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To facilitate further description of the embodiments of this disclosure, the following drawings are provided to illustrate and not to limit the scope of the disclosure.

[0018] Fig. 1A is a schematic showing the synthesis of PSC-conjugated panitumumab.

[0019] Fig. 1B is a schematic showing the radiolabeling of PSC-conjugated panitumumab with203Pb.

[0020] Fig. 1C shows the measurement of203Pb2+incorporation efficiency and radiochemical purity of purified203Pb-PSC-panitumumab by radio-TLC.

[0021] Fig. 1 D shows the measurement of203Pb2+incorporation efficiency and radiochemical labeling of purified203Pb-PSC-panitumumab by SDS-PAGE.

[0022] Fig. 2A shows the cellular uptake of203Pb-PSC-panitumumab in EGFR+ FaDu cells.

[0023] Fig. 2B shows the studies of203Pb-PSC-panitumumab binding competed with increasing amounts of unlabeled panitumumab.

[0024] Fig. 2C is a representative double inverse plot from Lindmo assay performed in FaDu cells with203Pb-PSC-panitumumab.DETAILED DESCRIPTION

[0025] As used herein, the terms “comprising”, “having”, “including”, and “containing” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps, even if a feature / component defined as a part thereof consists or consists essentially of specified feature(s) / component(s). The term “consisting essentially of” if used herein in connection with a compound, composition, use or method, denotes that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited compound, composition, method or use functions. The term “consisting of” if used herein in connection with a feature of a compound, composition, use or method, excludes the presence of additional elements and / or method steps in that feature. A compound, composition, use or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to. A use or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0026] A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictates otherwise. The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0027] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range including all whole numbers, all integers and, where suitable, all fractional intermediates (e.g., 1 to 5 may include 1 , 1.5, 2, 2.75, 3, 3.80, 4, and 5 etc.).

[0028] Unless otherwise specified, “certain embodiments”, “various embodiments”, “an embodiment” and similar terms includes the particular feature(s) described for that embodiment either alone or in combination with any other embodiment or embodiments described herein, whether or not the other embodiments are directly or indirectly referenced and regardless of whether the feature or embodiment is described in the context of a method, product, use, composition, compound, et cetera.

[0029] As used herein, the terms “treat”, “treatment”, “therapeutic” and the like include ameliorating symptoms, reducing disease progression, improving prognosis and reducing recurrence.

[0030] As used herein, the term “conjugate” refers to a compound having a binding moiety that is linked to a chelating moiety, such as a metal chelating moiety, either via direct bond or via a linker. For example, the chelating moiety may be a compound capable of chelating radionuclides, such as203Pb and / or212Pb. The binding moiety may be capable of selectively binding to a cellular target, and may for example be any of a peptide, protein, monoclonal antibody, polyclonal antibody, antibody fragment, or a synthetic protein. According to certain embodiments, a conjugate that includes an antibody or antibody fragment may be an “immunoconjugate”.

[0031] As used herein, the term “radiopharmaceutical” refers to a compound that is labeled with a radionuclide and is suitable for medical purposes, including treatment and diagnostic purposes, such as treatment of a disease state, including cancer (a radiotherapeutic), or diagnostic or imaging applications (a radiodiagnostic).

[0032] As used herein, the term “antibody” is used in the broadest sense and includes polyclonal antibodies and fragments thereof, monoclonal antibodies and fragments thereof, and includes intact antibodies and antibody fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific and trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di- scFv, tandem tri-scFv. As used herein, the “antibody fragment ” should be understood to encompass functional antibody fragments thereof, including functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragment. The term “antibody” also encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. For example, the antibody can be an antibody that is selective for epidermal growth factor receptor (EGFR), such as panitumumab, cetuximab, necitumumab, and amivantamab.

[0033] The term “amino acid residue” or “amino acid” includes reference to an amino acid that is incorporated into a protein, polypeptide, and / or peptide. The term “polypeptide” includes any polymer of amino acids or amino acid residues. The term “polypeptide sequence” refers to a series of amino acids or amino acid residues which physically comprise a polypeptide. A “protein” is a macromolecule comprising one or more polypeptides or polypeptide “chains,” such as for example a naturally occurring or synthetic protein. A “peptide” is a small polypeptide of a size of 2 to 20 amino acid residues. The term “amino acid sequence” refers to a series of amino acids or amino acid residues which physically comprise a peptide or polypeptide or protein depending on the length.

[0034] The terms “amino acid,” “amino acid residue,” “amino acid sequence,” or polypeptide sequence include naturally occurring amino acids (including L and D isosteriomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a similar manner as the common natural amino acids,such as selenocysteine, naphthylalanine, norleucine, pyrrolysine, N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine (see, e.g., Ho J et al., ACS Synth Biol 5: 163-71 (2016); Wang Y, Tsao M, Chembiochem 17: 2234-9 (2016)).

[0035] As used herein, the term “immunoconjugate” refers to a compound comprising at least one antigen binding region derived from an antibody (e.g., variable regions or complementarity determining regions) further coupled to at least one chelating moiety, such as the lead (Pb) specific chelator (PSC) described herein. The chelating moiety may, for example, be conjugated to one or more lysine or cysteine resides of an antibody or antibody fragment having the antigen binding region.

[0036] As used herein, “linker” refers to any chemical group that serves to couple a binding moiety to a chelating moiety, such as a lead specific chelating moiety. That is, the “linker” may correspond to the moiety “L” in any of the Formulas 1 a, 1 b, and 2b herein (“L” may also simply be a direct bond between the lead specific chelating moiety and the binding moiety A, in certain embodiments). For example, in certain embodiments, the linker can comprise an isothiocyanate group that is capable of forming a thiourea linkage with lysine residues present in a binding moiety corresponding to an antibody.

[0037] A binding moiety or conjugate having the binding moiety that “binds” a cellular target of interest is one that binds the cellular target with sufficient affinity that is measurably different from a non-specific interaction. Selective binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. “Selective” binding refers to binding moiety or conjugate comprising the binding moiety that is capable of binding a cellular target with sufficient affinity such that the binding moiety and / or conjugate is useful as a diagnostic and / or therapeutic agent with respect to the target. The “target” as used herein may be any biological target that is associated with a disease state, such as cancer, and may be for example be understood to be a protein, peptide, sequence, antigen, epitope, polysaccharide, lipid, or other biological structure that is associated with the disease state, such as forexample epidermal growth factor receptor (EGFR) that may be overexpressed in certain types of cancers. In one embodiment, the extent of binding of the binding moiety and / or conjugate to an unrelated target is less than about 10% of the binding of the binding moiety and / or conjugate to its target as measured, e.g., by a radioimmunoassay. A “target selective” binding moiety, as used herein, is one that specifically binds to the target with sufficient specificity and affinity to be useful in targeting a therapeutic, targeting a diagnostic, or method of detecting the target in a biological sample or a tissue from a subject. For example, an “antigen specific” antibody or immunoconjugate, as used herein, is one that specifically binds to the antigen with sufficient specificity and affinity to be useful in targeting a therapeutic, targeting a diagnostic, or method of detecting the antigen in a biological sample or a tissue from a subject. In some embodiments, binding moiety and / or conjugate, such as an immunoconjugate or antibody construct or target imaging complex or radioimmunoconjugate that binds to its target has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'10M, e.g., from 10’8M to 10'13M).

[0038] As used herein, the term “diagnostic agent” includes an “imaging agent”. As such, a “diagnostic radionuclide” includes radionuclides that are suitable for use in imaging agents.

[0039] The term “subject” refers to an animal (e.g. a mammal or a non-mammal animal). The subject may be a human or a non-human primate. The subject may be a laboratory mammal (e.g., mouse, rat, rabbit, hamster and the like). The subject may be an agricultural animal (e.g., equine, ovine, bovine, porcine, camelid and the like) or a domestic animal (e.g., canine, feline and the like). In some embodiments, the subject is a human.

[0040] The terms “cancer” and “cancerous” as used herein refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, melanoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g., epithelial squamous cell cancer), skin cancer, melanoma, lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high grade serous ovarian carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms’ tumor), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Additional examples of cancer include, without limitation, retinoblastoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including nonHodgkins lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi’s sarcoma, melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, anaplastic astrocytoma, basal cell carcinoma (basal cell epithelioma), bile duct cancer, small cell bladder cancer, metastatic breast cancer, metastatic colorectal cancer, epithelial ovarian cancer, fallopian tube cancer, gastric adenocarcinoma, glioblastoma multiforme (GBM), recurrent glioblastoma multiforme (GBM), gliomas, gliosarcoma, head and neck squamous cell carcinoma (HNSCC), recurrent head and neck squamous cell carcinoma, malignant pleural mesothelioma head and neck cancer, Hodgkin lymphoma, metastatic renal cell carcinoma, metastatic renal clear cell carcinoma, squamous nonsmall cell lung cancer, squamous carcinoma of the lung, relapsed or refractory small-cell lung cancer, treatment-resistant melanoma, metastatic melanoma, Merkel cell carcinoma, neuroendocrine cancer, large cell neuroendocrine cancer, neuroendocrinetumors (NETS), ovarian carcinoma, papillary carcinoma, peritoneal cancer, neuroendocrine prostate cancer, hormone- refractory prostate cancer, castrationresistant prostate cancer, soft tissue sarcoma, and squamous cell carcinoma.

[0041] As used herein, the term “epidermal growth factor receptor (EGFR)” refers to a type 1 membrane protein of 170 kDa, which is used interchangeably with an epithelial cell proliferation factor receptor or epidermal cell growth factor receptor, and is a cell surface receptor for extracellular protein ligands of the epidermal growth factor family, a subfamily of four closely related kinases, EGFR, HER2 / c-neu, Her 3, and Her 4. Activation of the receptor is known to be very important for the innate immune response in epidermal cells and known to be overexpressed in various kinds of tumors and / or cancers. For example, overexpression of EGFR is observed in cancers of lung cancer, breast cancer, colon cancer, stomach cancer, brain cancer, bladder cancer, head cancer, neck cancer, ovarian cancer, and prostate cancer. Tumor cells in which EGFR is overexpressed may produce epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) which are ligands of EGFR. In this regard, the ligands bind to EGFR to induce cell proliferation and tumor growth. For example, EGFR mRNA and protein are overexpressed in about 40-90% of head and neck cancer. As used herein, “EGFR+” or “EGFR positive” refers to cells or tissues that express EGFR. EGFR+ tumors or EGFR+ cancers refer to tumors or cancers express EGFR.

[0042] As used herein, the terms “salt” and “solvate” have their usual meaning in chemistry. As such, when the compound is a salt or solvate, it is associated with a suitable counter-ion. It is well known in the art how to prepare salts or to exchange counter-ions. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of a suitable base (e.g. without limitation, Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are generally carried out in water or in an organic solvent, or in a mixture of the two. Counter-ions may be changed, for example, by ion-exchange techniques such as ion-exchange chromatography. All zwitterions, salts, solvates and counter-ions are intended, unless a particular form is specifically indicated.

[0043] In certain embodiments, the salt or counter-ion may be pharmaceutically acceptable, for administration to a subject. As used herein, “pharmaceutically acceptable” means suitable for in vivo use in a subject, and is not necessarily restricted to therapeutic use, but also includes diagnostic use. More generally, with respect to any pharmaceutical composition disclosed herein, non-limiting examples of suitable excipients include any suitable buffers, stabilizing agents, salts, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, or polymers for sustained or controlled release. See, for example, Berge et al. 1977. (J. Pharm Sci. 66:1 -19), or Remington-The Science and Practice of Pharmacy, 21 st edition (Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia), each of which is incorporated by reference in its entirety.

[0044] As used herein, the term “alkyl group” encompasses saturated linear or branched carbon radicals having, for example, one to about twenty carbon atoms or, in specific embodiments, one to about twelve carbon atoms. In other embodiments, alkyl groups are "lower alkyl" groups having one to about six carbon atoms. Examples of such groups include, but are not limited thereto, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, ethylhexyl, octyl and the like.

[0045] As used herein, the term “radionuclide” or “radioisotope” includes, but is not limited to, an alpha emitting radionuclides, beta-emitting radionuclides, and / or gamma-emitting radionuclides, such as, e.g., any one of Y-86, Y-90, Lu-177, In-111 , Re-186, Re-188, Sr-89, Sm-153, Ac-225, Bi-213, Po-213, Bi-212, Ra-223, Ra-224, Tc- 99m, Th-227, Tb-149, Tb-161 , Ga-68, Cu-64, Cu-67, Zr-89, Cs-137, Pb-203, Pb-212, and Pd-103, among others. For example, lead radionuclides can include any of Pb-196, Pb-197, Pb-198, Pb-199, Pb-200, Pb-201 , Pb-202, Pb-203, Pb-204, Pb-205, Pb-206, Pb-207, Pb-208, Pb-209, Pb-210, Pb- 211 , Pb-212, Pb-213, Pb-214, Pb-215, and Pb- 216. As used herein, the expressions “XE” and “E-x”, where “E” represents the element and “x” represents the particular elemental isotope, are equivalent and have the same meaning. For example, “xPb” and “Pb-x” (and “lead-x”) are equivalent and have thesame meaning, such that “203Pb” is equivalent to “Pb-203” (and to “lead-203”), and “212Pb” is equivalent to “Pb-212” (and “lead-212”).

[0046] As used herein, lead (Pb) refers to the lead element and includes isotopes of both radioactive and observationally stable. The isotopes of Pb include, but are not limited to, Pb-196, Pb-197, Pb-198, Pb-199, Pb-200, Pb-201 , Pb-202, Pb-203, Pb-204, Pb-205, Pb-206, Pb-207, Pb-208, Pb-209, Pb-210, Pb- 211 , Pb-212, Pb-213, Pb-214, Pb-215, and Pb-216.

[0047] Targeting vectors in radiotheranostics for TRT encompass small molecules, peptides, antibodies, and nanoparticles, among other things (4). Among the targeting vector landscape, monoclonal antibodies (mAb) display exceptional target specificity, making mAb excellent candidates for TRT.

[0048] Panitumumab is an FDA-approved human monoclonal antibody specific to EGFR used as a single drug or in combination with other drugs to treat certain types of colorectal cancer (CRC), especially for the treatment of metastatic colorectal carcinoma with disease progression. Panitumumab binds to the extracellular domain of the EGFR, preventing EGFR dimerization and, thus, halting ligand-induced receptor autophosphorylation and intracellular signalling pathway activation (18). EGFR is overexpressed in many solid tumour cancers, including CRC, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and breast cancer. (19,20). Several studies demonstrated the relationship between EGFR overexpression and survival rate in these cancers: as EGFR expression increased, survival decreased (21 ), making EGFR a promising target for TRT of solid tumours.

[0049] According to aspects of the present disclosure, panitumumab was successfully modified with PSC-NCS as a novel Pb-specific bifunctional chelating agent. PSC-decorated human antibody panitumumab was rapidly and reproducibly radiolabeled with203Pb(OAc)2 in good radiochemical yields under mild reaction conditions compatible with the structural and functional integrity of an antibody. Novel immunoSPECT probe203Pb-PSC-panitumumab showed EGFR-mediated uptake in FaDu cells. The present disclosure introduces203Pb-PSC-panitumumab as a novel immuno-SPECT probe for imaging EGFR+ tumours and an opportunity to develop203 / 21 spb-psC-panitumumab radiotheranostics for combined SPECT imaging and targeted alpha therapy of EGFR-expressing cancers.Conjugate

[0050] Accordingly, one aspect of the present disclosure is directed to a conjugate for radiotheranostics. Generally speaking, the conjugate comprises a binding moiety A that is bound to a lead specific chelating moiety, either via a direct bond or via a linker, having the formula A-(L-C)n, where n is at least 1 , A is the binding moiety, L is a bond or linker, and C is a lead specific chelating moiety. According to one embodiment, the lead specific chelating moiety C has a formula according to Formula 5a:Formula 5a where in Formula 5a, each Ri is independently hydrogen or an alkyl group,R2 is -ORi, -NFh or a bond connecting the lead specific chelating moiety, C, to the linker, L,R4 and Rs are independently hydrogen or a bond connecting the lead specific chelating moiety to the linker, L, provided (i) at least one of R4 and Rs is hydrogen, and (ii) each of R4 and Rs is hydrogen when R2 is a bond connecting the lead specific chelating moiety, C, to the linker, L, and n is at least 1 .

[0051] In one embodiment, in a case where the other OR1 groups in Formula 5a are each OH, R2 is other than an OH group, such as -NH2, -OR1 where R1 is an alkyl group, or a bond connecting the lead specific chelating moiety, C, to the linker, L.

[0052] According to one embodiment, a conjugate according to the present disclosure is shown in any of Formulas 1 a, 1 b and 1 c below:Formula 1aFormula 1c

[0053] In Formulas 1a, 1b and 1c,

[0054] A is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR),L is a bond or a linker,R3 is -OR1 or -NH2, wherein R1 is an alkyl group, and n is at least 1 .

[0055] The chelating moiety in Formulas 5a and 1a-1c is a lead-specific chelating moiety derived from 1 ,4,7,10-tetraazacyclododecane-7-acetamide-1 ,4,10-triacetic acid (lead specific chelator (PSC)), as described in further detail below. Furthermore, the binding moiety A is one that is selective for epidermal growth factor receptor (EGFR) and may be any selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein. For example, in one embodiment, the binding moiety A is the monoclonal antibody panitumumab. The number of chelating moieties bound to the antibody A via L (which is a bond or linker) is at least 1 , but may be at least 2, at least 3, at least 4, at least 6, at least 7, at least 8 and / or at least 10 (e.g., n in Formula 1a above is an integer between 1 -20, such as between 1-8). In one embodiment, the average number of chelating moieties bound to the antibody A in a formulation containing the conjugate is in the range of 3-5 (e.g., n is from 3-5, such as from 4-5). In another embodiment, the average number of chelating moieties bound to the antibody A in a formulation containing the conjugate is in the range of 3-20 (e.g., n is from 5-15, such as from 7-12).

[0056] According to one embodiment, the chelating moiety in any of Formulas 5a and 1a-1c is bound to the binding moiety A via a linker L comprising a thiourea linkage, as shown for example in Formula 2a, 2b and 2c below, where n is any integer between 1 -20, such as between 1-8, as described above. In Formula 2a, 2b and 2c, Z is -NH- and is the residue of a primary amine moiety comprised by A, which NH group is contributed by the binding moiety A to participate in the thiourea linkage. As shown, the binding moiety may contribute a plurality of NH groups to form thiourea linkages with the linker L, in a case where n is greater than 1.

[0057] The conjugate A-(L-C)n, such as any of the conjugates according to any of Formulas 5a, 1 a-1 c and / or 2a-2c may be further labeled, such as with a lead (Pb) isotope, and specifically with a lead radioisotope, to provide a theranostic compound that can be used for diagnostic and / or therapeutic purposes. Examples of the labeled conjugates are shown in Formulas 1a-1 , 2a-1 , 2b-1 and 2c-1 below.

[0058] In Formula 1a-1 above,xPb is a lead isotope, n is at least 1 , A is the binding moiety, and L is a bond or linker. The corresponding Formulas 1 b and 1 c shown above can be similarly labeled with a lead isotope, analogously to the structure shown in Formula 1a-1.Formula 2a-1

[0059] In Formula 2a-1 above, A is the binding moiety, the adjacent -NH- group is the residue of a primary amine moiety comprised by A, n is at least 1 , andxPb is an isotope of lead.Formula 2b-1 n

[0060] In Formula 2b-1 above, A is the binding moiety, the adjacent -NH- group is the residue of a primary amine moiety comprised by A, n is at least 1 , andxPb is an isotope of lead.

[0061] In Formula 2c-1 , A is the binding moiety, the adjacent -NH- group is the residue of a primary amine moiety comprised by A, n is at least 1 , andxPb is an isotope of lead.

[0062] Further disclosure of the conjugate, compounds, formulations, and methods therewith, are disclosed below.Chelating Moiety

[0063] The chelating moiety used to prepare the conjugate described herein is a lead specific chelating moiety that is based on the lead specific chelator (PSC) 2,2’- (4, 10-bis(2-amino-2-oxoethyl)-1 ,4,7, 10-tetraazacyclododecane-1 , 7-d iy l)d iacetic acid compound shown in Formula 4 below.

[0064] The chelating moiety can be modified and / or derivatized to link to the binding moiety A, such as by connecting the chelating moiety at any one or more of the locations *, ** or *** in the Formula 5b below.Formula 5b

[0065] In Formula 5b above, each Ri may independently be hydrogen, an alkyl group, or a protecting group, such as a tert-butyl group. R2 in Formula 5b can be OR1 or can be NH2. For example, tert-butyl groups may be provided as protecting groups during synthesis of the derivatized chelating moiety and / or conjugate, and may be removed at an appropriate point during synthesis or after forming the final conjugate. In one embodiment, in a case where the other OR1 groups in Formula 5b are each OH, R2 is other than an OH group, such as NH2 or OR1 where R1 is an alkyl group. According to certain embodiments, for example, a derivatized PSC can be prepared from a 1 ,4,7,10-tetraacecyclododecane-1 ,7-bis(t-butyl acetate) precursor via a monoamide(43).

[0066] According to certain embodiments, the lead specific chelator (PSC) can be derivatized to add an isothiocyanate moiety (NCS) that is linked to the chelator through a benzyl group, as shown for example in Formula 3a below (as connected at point * in Formula 5b):

[0067] Formulas 3b and 3c further depict the chelator (PSC) as derivatized at the connection points ** and ***, respectively, in Formula 5b.

[0068] In each of Formulas 3a-3c above, each Ri may independently be hydrogen, an alkyl group, or a protecting group, such as a tert-butyl group, as described for Formula 5a above. Accordingly, in some embodiments of the present disclosure, the derivatized chelator that is used to prepare the conjugate is PSC is 2,2'-(4-(2-amino- 2-oxoethyl)-10-(2-((4-isothiocyanatobenzyl)amino)-2-oxoethyl)-1 ,4,7,10 tetraazacyclododecane-1 ,7-diyl) diacetic acid (PSC-NCS), corresponding to Formula 3a above, where each Ri is hydrogen (11 ). Other derivatized lead-specific chelating moieties may also be used.

[0069] According to certain embodiments, the isothiocyanate group of the derivatized lead-specific chelators of any of Formulas 3a-3c may be reacted with a suitable site on the binding moiety A, to form a thiourea linkage with the binding moiety. For example, the isothiocyanate group may react with a lysine residue, or other suitable residue (e.g. a residue having an amine group), that may be present in the binding moiety A. Furthermore, as described above, in certain embodiments a plurality ofchelating moieties may be linked to the binding moiety A, such as for example by reaction of a plurality of NCS-derivatized lead specific chelators with a plurality of lysine residues in the binding moiety A. Formulas 2a-2c below show general formulas for a conjugate having a moiety A which is bound to at least one, or a plurality, of lead specific chelating moieties, via a thiourea linkage formed via reaction of a primary amine moiety comprised by binding moiety A with an isothiocyanate group on the PSC-NCS derivatized lead-specific chelator of any of Formulas 3a, 3b and 3c. As shown in Formulas 2a-2c below, Z is -NH- and is the residue of a primary amine moiety comprised by A, and participates in the thiourea linkage, to link the chelating moiety to the binding moiety A via the thiourea group.

[0070] In Formulas 2a-2c above, A is the binding moiety, n is at least one, as described above, and Z is -NH- and is the residue of a primary amine moiety comprised by A.Radionuclide

[0071] The lead specific chelating moiety is capable of chelating a lead isotope, such as a lead radionuclide. The conjugate having the chelated lead radionuclide may be suitable for theranostic methods, such as imaging and diagnosis, as well as therapeutic treatments. Lead isotopes that the lead specific chelating moiety may be capable of chelating can include both radioactive (radionuclide) and non-radioactive isotopes, for example, any of Pb-196, Pb-197, Pb-198, Pb-199, Pb-200, Pb-201 , Pb- 202, Pb-203, Pb-204, Pb-205, Pb-206, Pb-207, Pb-208, Pb-209, Pb-210, Pb- 211 , Pb- 212, Pb-213, Pb-214, Pb-215, and Pb-216. Of these, the lead radionuclides (e.g. radioactive lead isotopes) that the lead specific chelating moiety is capable of chelating include any selected from the group consisting of Pb-196, Pb-197, Pb-198, Pb-199, Pb- 200, Pb-201 , Pb-202, Pb-203, Pb-205, Pb-209, Pb-210, Pb- 211 , Pb-212, Pb-213, Pb- 214, Pb-215, and Pb-216. According to certain embodiments, the lead radionuclide that is conjugated by the lead specific chelating moiety comprises any of Pb-203 or Pb-212. Pb-203 emits y-photons through electron capture, allowing detection with single-photon emission computed tomography (SPECT) for diagnostic imaging, whereas Pb-212 decays by emitting |3’-particles and a-particles suitable for delivering therapeutic doses of radiation to cancer cells (13,14). Accordingly, in one embodiment, the conjugate comprises a lead specific chelating moiety that is labeled with lead-203, for diagnostic and / or imaging applications. In another embodiment, the conjugate comprises a lead specific chelating moiety that is labeled with lead-212, for therapeutic treatment applications, such as in the treatment of cancer.

[0072] Formulas 1 a-1 , 2a-1 , 2b-1 and 2c-1 below show: the general conjugate of Formula 1 a containing the lead specific chelating moiety as radiolabeled with a lead isotope such as a lead radionuclide (Formula 1 a-1 ); and the more specific conjugate (Formulas 2a, 2b and 2c) containing the lead specific chelating moiety that is linked viaa thiourea group to the binding moiety A (Formula 2a-1 , 2b-1 , 2c-1 ), where x may represent any of the lead isotopes or radionuclides herein, such as for example 203 for203Pb, or 212 for212Pb.

[0073] More generally, the conjugate A-(L-C)n, where C is the chelating moiety in Formula 5a above, can be radiolabeled with a lead radioisotopexPb, such as for example203Pb, or212Pb.Binding Moietv / EGFR

[0074] According to certain embodiments, the binding moiety A may be any of a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein, that is selective for epidermal growth factor receptor (EGFR).

[0075] Epidermal growth factor receptor (EGFR) refers to a type 1 membrane protein of 170 kDa, which is used interchangeably with an epithelial cell proliferation factor receptor or epidermal cell growth factor receptor, and is a cell surface receptor for extracellular protein ligands of the epidermal growth factor family, a subfamily of four closely related kinases, EGFR, HER2 / c-neu, Her 3, and Her 4. Activation of the receptor is known to be very important for the innate immune response in epidermal cells and known to be overexpressed in various kinds of tumors. For example, overexpression of EGFR is observed in cancers of lung cancer, breast cancer, colon cancer, stomach cancer, brain cancer, bladder cancer, head cancer, neck cancer, ovarian cancer, and prostate cancer. Tumor cells in which EGFR is overexpressed may produce epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) which are ligands of EGFR. In particular, EGFR is overexpressed in many solid tumour cancers, including colorectal carcinoma (CRC), head and neck squamous cell carcinoma (HNSCC), nonsmall cell lung cancer (NSCLC) and breast cancer. (19,20). Several studies demonstrated the relationship between EGFR overexpression and survival rate in these cancers: as EGFR expression increased, survival decreased (21 ), making EGFR a promising target for TRT of solid tumours.

[0076] Specifically, EGFR+ in HNSCC accounts for almost 90% of HNSCC cases diagnosed in the clinic (26). This understanding offers EGFR as a suitable target receptor to target head and neck cancer cells when developing radiotheranostics for HNCC. Creating a radiotheranostic pair using radionuclides of the same chemicalelement that targets EGFR provides a probe for imaging and treatment with similar pharmacokinetics, leading to similar biodistribution in EGFR+ tumours (1 ,8).

[0077] According to one embodiment, the binding moiety comprises an antibody or antibody fragment having selectivity for EGFR, such as for example any one or more of cetuximab, panitumumab, amivantamab and necitumumab, and / or any of the further anti-EGFR antibodies listed in Table 1 below. Other antibodies, antibody fragments, or synthetic proteins, such as antibody derivatives, may also be used. In certain embodiments, the anti-EGFR antibody is a full-length immunoglobulin. In certain embodiments, the ant-EGFR antibody is an engineered antibody fragment such as a single-chain fragment variable (scFv), fragment antigen-binding region (Fab). In certain embodiments, the anti-EGFR antibody is a bi-specific, tri-specific, or multi-specific antibody which targets different epitopes of the EGFR protein. In certain embodiments, the anti-EGFR antibody is a bi-specific, tri-specific, or multi-specific antibody which targets EGFR and other cell membrane proteins. Unlimited examples of other cell membrane proteins can comprise epidermal growth factor (EGF), hepatocyte growth factor (FGF), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Delta-like ligand 3 (DLL3), fibroblast activation protein (FAP), PD-1 , PD-L1 , CTLA4, B7-H3, and mesenchymal-epithelial transition factor (MET). A person skilled in the art can use the methods disclosed herein to make conjugates of PSC with other EGFR antibodies, including those listed in Table 1 .Table 1Anti-EGFR Antibodies

[0078] In one embodiment, the binding moiety is panitumumab (also called Vectibix), which is a human monoclonal antibody EGFR antagonist. Panitumumab is currently indicated as a single agent for the treatment of metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine, oxaliplatin, and irinotecan chemotherapy regimens. Panitumumab is believed to bind to the extracellular domain of EGFR to prevent its activation. Panitumumab was FDA approved in September 2006 for the treatment of EGFR-expressing metastatic colorectal cancer (“Panitumumab (Vectibix)” by Gemmete et al., AJNR Am J Neuroradiol. 2011 Jun-Jul: 32(6) 1002-1003.)

[0079] Furthermore, according to certain embodiments, in case where the binding moiety comprises an antibody, the conjugate can comprise one or more chelating moieties bound to the antibody, either directly or through a linker, on both heavy and light chains of the antibody, or on just one of the heavy and / or light chains.

[0080] According to one embodiment, the binding moiety comprises the monoclonal antibody panitumumab, which is selective for EGFR, to facilitate targeting of cancerous cells that overexpress EGFR.Linker

[0081] The linker may be any chemical group that serves to couple the binding moiety A to the lead specific chelating moiety. That is, according to certain embodiments, the moiety “L” in any of the Formulas herein may be a bond that directly connects, or a linker that indirectly connects, the lead specific chelating moiety to the binding moiety A. For example, in certain embodiments, the linker can form a thiourea group that forms a thiourea linkage with primary amine moieties such as lysine residues present in a binding moiety A, for example as shown in Formulas 2a-2c above. For example, the linker can comprise the molecular structure shown in Formula 6 below, where the asterisks (*) indicate points of attachment to the lead specific chelating moiety on one end, and the binding moiety A on the other end (e.g. via a thiourea linkage):Formula 6

[0082] Notably, in Formula 2a shown above, “L” comprises both the moiety shown in Formula 6 as well as an NH group connecting the moiety shown in Formula 6 to the chelating moiety.

[0083] In addition to a thiourea linkage, the linker may comprise a moiety that is capable of forming a bond with the binding moiety A via a different type of linkage, such as by amide bond, disulfide bond, or other connection.

[0084] In certain embodiments, the linker is a hydrophobic linker consisting of an aliphatic carbon chain that connects the chelating moiety to the binding moiety. In certain embodiments, L is a hydrophilic linker that includes heteroatom substitutions in the aliphatic chain that connects the chelating to the binding moiety. In certain embodiments, L is a mixture of hydrophilic and hydrophobic entities including piperidine insertions of amino acid insertions to lengthen the chain and modulate the pharmacodynamics properties of the composition. In certain embodiments, L is PEGn, wherein n is 1 -10 (PEG is polyethylene glycol). In certain embodiments, n is 2, 4 or 8 PEG subunits. In certain embodiments, n is 4. In certain embodiments, L is an aliphatic (ALP) linker of 2 or 4 carbons. In certain embodiments, L is a piperidine (PIP) based linker.Method of Manufacture of Radiolabeled Compound

[0085] Previous reports for radiolabeling panitumumab with SPECT-detectable radioisotopes were labelling with111In (27,28) and177Lu (28); also, the Fab fragment of panitumumab was labelled with177Lu (29) and99mTc (30); however, no previous studies describing labelling panitumumab with203Pb have reported. Antibody-based compounds such as trastuzumab (Herceptin) via DOTA (31 ), c8C3 via TCMC (32), and peptides such as a-melanocyte-stimulating hormone (33), and low molecular weight PSMA ligands (34) have been labelled with203Pb using DOTA coordination chemistry. Also, the conditions required for labelling panitumumab with111In using diethylenetriaminepentaacetic acid (DTPA) and / or89Zr using p-isothiocyanatobenzyl-desferrioxamine B included heating to 37 °C, and depending on the chelating moieties administered, different reaction times of up to 4 h were required (35,36). Moreover, high temperatures (60-75°C) were reported for the radiolabeling of peptides with203Pb to achieve high incorporation efficiency with shorter incubation times (37,38).

[0086] According to one embodiment, a process for a preparation of the conjugate comprises reacting a binding moiety A that is selective for EGFR, such as a monoclonal antibody, polyclonal antibody, antibody fragment, or synthetic protein, with a lead specific chelating moiety, such as for example via one or more linkers present on either or both of the binding moiety and / or lead specific chelating moiety, or via direct binding of the chelating moiety to the binding moiety. For example, the lead specific chelating moiety may be a derivatized chelator according to any of Formulas 3a-3c above, having an isothiocyanate group that is capable of reacting with lysine residues (or other primary amine-containing residues) in the binding moiety. According to one embodiment, the derivatized chelator of Formula 3a, where Ri is hydrogen, is reacted with panitumumab, to prepare the conjugate. According to yet further embodiments, the binding moiety A (e.g. panitumumab), can be combined with the lead specific chelating moiety to provide a conjugate having a ratio of binding moiety A to lead specific chelating moiety of 1 :1 , 1 :2, 1 :2, 1 :4, 1 :5, 1 :6, 1 :7, and / or 1 :8, or in other words to provide multiple lead specific chelating moieties bound to the binding moiety A in the conjugate.

[0087] According to certain embodiments, the conjugate can be radiolabeled, such as with a lead radionuclide, to provide a radiotherapeutic and / or radiodiagnostic molecule (i.e., a radiopharmaceutical).

[0088] According to certain embodiments, the radiolabeling of the conjugate with lead, such as203Pb and / or212Pb, can occur at temperatures close to room temperature (25°C), and require a relatively short period of time to achieve desired levels of radiolabeling. According to one embodiment, the conjugate is combined with the lead radioisotope for radiolabeling at a temperature of no more than 60°C, no more than 55°C, no more than 50°C, no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C, and / or no more than 20°C. For example, the conjugate may be combined with the lead radioisotope for radiolabeling at a temperature that is in the range of from 20°C to 60°C, 20°C to 40°C, 20°C to 30°C, and / or about room temperature (25°C). According to one embodiment, the conjugate is combined with the lead radioisotope for radiolabeling for no more than 60 minutes, no more than 30 minutes, no more than 20 mins, no more than 15 mins, and / or no morethan 10 mins, and at least 1 min, at least 3 mins, at least 4 mins, and / or at least 5 mins. Furthermore, according to certain embodiment, the conjugate is combined with the lead radioisotope for radiolabeling in an aqueous solution having a pH in a range of from 4 to 6, and / or a pH of about 5.

[0089] For example, according to certain aspects of the present disclosure, radiolabeling of PSC-panitumumab may be performed at a pH of 5.0 and about room temperature (25°C) for 5-10 minutes with [203Pb]Pb(OAc)2, and the incorporation efficiency can be determined using radio-TLC. Specifically, according to certain embodiments, an average of 4 to 5 Pb specific chelator (PSC) chelators (chelating moieties) can be attached to one antibody, with radiolabeling efficiency was 99.2±0.7%. According to certain embodiments, isolated radiochemical yield of203Pb-PSC- panitumumab was 41 .4 ± 8 %, and the molar activity was 1 .2 ± 0.35 GBq / mg.

[0090] According to certain aspects of the present disclosure, the labeling process with the derivatized PSC chelator does not require any elevated temperatures, such as above 60°C, or even above 30°C and / or 25°C, and203Pb or212Pb incorporation can proceed with high efficiency (>99%) at short reaction times of 5-10 min at about room temperature (25°C).Diagnostic and Therapeutic Uses

[0091] Embodiments of the present disclosure further provide methods for imaging or diagnosing a cancer or tumor in a subject in need thereof, by administering a radiopharmaceutical comprising a radiolabeled conjugate described herein to the subject, and imaging tissue having the radiolabeled conjugate bound thereto. That is, when the radiolabeled conjugate is labeled with a lead radioisotope such as203Pb (Pb- 203) that is suitable for imaging purposes, the conjugate can be administered to a subject and will accumulate at target sites for which the binding moiety is selective. Imaging techniques such as SPECT imaging can be performed to view the accumulation of the radiolabeled conjugate at target sites, which may be indicative of a location of cancerous cells, such as cancerous cells overexpressing EGFR.

[0092] According to certain aspects,203Pb (Pb-203) may be suitable for imaging processes such as SPECT, to provide for imaging and diagnosing of the presence or extent of cancer and / or tumors in a subject.

[0093] Furthermore, according to certain embodiments, a method of treatment of cancer can be provided in a subject in need thereof, by administering a radiopharmaceutical comprising a radiolabeled conjugate described herein to the subject, in therapeutically effective amounts that are sufficient to treat the subject. That is, when the radiolabeled conjugate is labeled with a lead radioisotope such as212Pb (Pb-212) that is suitable for treatment purposes, the conjugate can be administered to a subject and will accumulate at target sites for which the binding moiety is selective, and which may be indicative of a location of cancerous cells, such as cancerous cells overexpressing EGFR. The radiation emitted by the radiolabeled conjugate bound to the target site may provide a cell killing effect that is selective for the target cells, thereby providing treatment. According to certain embodiments, the cancer is associated with EGFR overexpression in the subject. In some embodiments, the cancer is selected from the group consisting of colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), and breast cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC).

[0094] Aspects of the present disclosure further provide203 / 212Pb-PSC- panitumumab as ideal radiotheranostics for combined SPECT imaging and targeted alpha therapy of EGFR-expressing cancers.203 / 212Pb radiotheranostics represent an attractive alternative to currently used225Ac-based radiotheranostics for targeted alpha therapy. The availability of212Pb through the emerging224Ra / 212Pb generator technology and the ideal radionuclide matching pair characteristics of radiometals203Pb and212Pb ensure identical coordination chemistry and identical biodistributions profiles of203 / 212Pb radiotheranostics. This represents a significant advantage to currently used225Ac-based radiotheranostics for targeted alpha therapy relying on chemically different imaging surrogates, such as133La and134Ce (42).

[0095] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.Formulations

[0096] The radiolabeled conjugates / radiopharmaceutical compounds described herein can be administered in a radiopharmaceutical formulation suitable for delivery to a subject, such as for example via intravenous administration, and may be in form of an aqueous solution (e.g. saline solution) containing the radiolabeled conjugate in a concentration sufficient for one or more of diagnostic and therapeutic purposes, as well as optional stabilizers and / or oxidants, buffers, etc. According to one embodiment, a suitable dosage of the radiolabeled conjugate, such as for example the radiolabeled conjugate according to any of Formulas 2a-1 , 2b-1 or 2c-1 above being used for diagnostic or medical imaging purposes, where the binding moiety is panitumumab, and the lead radionuclide is203Pb (Pb-203), may be from 50-500 MBq, preferably between 100-400, and more preferably between 200-300 MBq. According to one embodiment, a suitable dosage of the radiolabeled conjugate, such as for example the radiolabeled conjugate according to any of Formulas 2a-1 , 2b-1 or 2c-1 above being used for therapeutic treatment purposes, where the binding moiety is panitumumab, and the lead radionuclide is2212Pb (Pb-212), may be from 18-555 MBq, preferably between 37-370, and more preferably between 74-185 MBq. The radiolabeled conjugate may be prepared in phosphate-buffered saline (PBS) with or without stabilizers including any one or more of ascorbic acid, sodium acetate, gentisic acid, a chelator (e.g. DTPA (diethylenetriaminepentaacetic acid) or DSMA (dimercaptosuccinic acid)), and ethanol, at concentrations that are suitable for human or animal use.EXAMPLESExample 1 Production of203Pb

[0097] 203Pb was produced using a recently published procedure (12). Briefly,205TI metal (99.9% isotopic enrichment) targets were irradiated at 23.3 MeV on a TR-24 cyclotron at currents up to 60 pA to produce203Pb via the205TI(p,3n)203Pb nuclear reaction. Following a cool-down period of >12 hours, targets were removed and irradiated205TI dissolved in 4 M HNO3. A NEPTIS Mosaic-LC synthesis unit performed automated separation using Eichrom Pb resin, and203Pb was eluted using 8 M HCI or 1 M NH4OAC. Purified203Pb yields of up to 12 GBq were attained (15.8 GBq at EOB). The [203Pb]PbCl2 and [203Pb]Pb(OAc)2 products contained no detectable radionuclidic impurities besides201Pb (<0.1 %), and <0.4 ppm stable Pb.205TI metal was recovered with a 92% batch yield.Example 2 Preparation of203Pb-PSC-panitumumab radioimmunoconjugateGeneral

[0098] All glassware was rinsed with ultra-pure HCI (Fisherbrand, A508-P500). Trace metal-based ultra-pure chemicals for buffer preparations were purchased from Sigma Aldrich. All buffer solutions were treated with biotechnology-grade Chelex 100 (Bio-Rad, 143-2832).PSC functionalization of panitumumab and radiolabeling with [203Pb]Pb(OAc)2

[0099] 2,2'-(4-(2-amino-2-oxoethyl)-10-(2-((4-isothiocyanatobenzyl)amino)-2- oxoethyl)-1 ,4,7,10-tetra-azacyclododecane-1 ,7-diyl) diacetic acid (PSC-NCS) chelator (200 pg) was dissolved in 50 pL of 0.1 M NaHCOs (pH = 9.0) and added to 400 pL panitumumab (Vectibex ® 20 mg / mL). The pH was adjusted to 8.0 and left on a thermoshaker set at 800 rpm and 30 °C for 2.5 hours.

[0100] The samples were purified via size exclusion chromatography (Bio-Rad 10DG desalting column, USA), which was pre-equilibrated and eluted with 0.025 M NaOAc buffer (pH=5.5). The antibody concentration of each fraction was measured using nanodrop (Thermo Scientific, NanoDrop OneC), and the fraction with the highest concentration was submitted for matrix-assisted laser desorption / ionization (MALDI) toassess the number of PCS chelators per antibody (~5 chelators per antibody). [203Pb]Pb(OAc)2 (150-200 MBq) was added to PSC-panitumumab (200 pg), and the reaction was kept at room temperature (about 25°C) for 5-10 minutes at pH=5.

[0101] Radio-thin layer chromatography (radio-TLC) analysis (AR-2000, Eckert and Ziegler) was used to determine203Pb incorporation efficiency by spotting samples on silica plates and using 20 mM EDTA and 0.2 M NaOAc as the mobile phase; in this system, the Rf for [203Pb]Pb(OAc)2 was 1.0 and for203Pb-PSC-panitumumab was zero (14).203Pb-PSC-panitumumab was purified on an Econo-Pac 10DG desalting column pre-equilibrated with 0.25 M sodium acetate, pH 5.5 used as the eluant.

[0102] Elution fractions (300 pL) were collected from the column, and the radioactivity was measured using an Atomlab 400 dose calibrator (Biodex, Shirley, NY, USA). Laemmli buffer (Bio-Rad, USA) was added to203Pb-PSC-panitumumab, and the samples (15 pL) were incubated at 95 °C for 5 minutes. Then, the samples were loaded on SDS-PAGE (Bio-Rad, Mini-PROTEAN® TGXTMPrecast Protein Gels) and ran at 120 V. The gel was imprinted on film and evaluated by autoradiography on a BAS-5000 phosphor imager (Fujifilm).Example 3 Functional characterization of203Pb-PSC-panitumumabMethodsCell uptake studies

[0103] EGFR-expressing neck and neck cancer FaDu cells were cultured in a 5% CO2 incubator at 37 °C in Gibco DMEM media supplemented with 10% fetal bovine serum (Gibco, USA) and 1 % penicillin / streptomycin with media renewal 2-3 times per week. For cell uptake studies, the cells were seeded in 6-well plates and left in the incubator overnight. The media was removed, and 500 pL of Kerbs-Ringer buffer solution (22) was added to each well, followed by the addition of 0.2 MBq of203Pb-PSC- panitumumab, and the plate was kept in the incubator at 37 °C at 5% CO2. Cell uptake was terminated at 1 , 5, 15, 30, 45, 60, and 90 min by adding ice-cold Krebs buffer andrinsing the wells twice to wash away unbound radioimmunoconjugates before lysing the cells with RIPA buffer. The cell lysates were transferred to scintillation vials and measured for radioactivity using a y-counter (Wizard2® 2480 Automatic Gamma Counter, Perkin-Elmer, Canada). According to the manufacturer's recommendations, protein levels were quantified using a Pierce™ BCA protein assay kit. For blocking studies, the FaDu cells were co-treated with different amounts of unlabeled panitumumab (0.5-10 mg) and 0.2 MBq of203Pb-PSC-panitumumab. Cell uptake levels were normalized to percent of the total amount of radioactivity per milligram of protein (% radioactivity / mg protein) and plotted as a function of time. All experiments were performed in triplicates.Immunoreactivity

[0104] The immunoreactivity of203Pb-PSC-panitumumab was assessed by a cell binding assay with EGFR-expressing FaDu cells using the Lindmo et al. method (23). FaDu cell concentrations of 0.25, 0.5, 1 , 2, 3, 4, and 5 x106were prepared in 500 pL of PBS and 1 % bovine serum albumin (Sigma, USA). Then, 20,000 cpm of203Pb-PSC- panitumumab was added to each cell concentration. The samples were placed on a rocker for one hour at room temperature (about 25°C). After triplicate PBS washes, the samples were transferred to 1.5mL LoBind® tubes (Eppendorf, USA), and by using a y- counter (Wizard2® 2480 Automatic Gamma Counter, Perkin-Elmer, Canada), the counts of each sample were determined.

[0105] The radioactivity data obtained was corrected in the background and compared to counts from the total activity added to control samples in the experiments. The immunoreactive fraction was calculated by performing a linear regression analysis on a double-inverse plot of (total / bound) activity versus normalized cell concentration.Example 4 Radiochemistry

[0106] MALDI analysis confirmed the conjugation of 4-5 PSC chelators per antibody by reacting the isothiocyanate group in PSC-NCS with lysine residues inpanitumumab (Figure 1A). PSC-conjugated panitumumab was used for radiolabeling with [203Pb]Pb(OAc)2, and the radiolabelling efficiency was measured with radio-TLC, indicating 99.2±0.7% incorporation of203pb2+(Figure 1 B and 1 C) which is comparable to results obtained by Nelson et al. (12). SDS-PAGE analysis further confirmed incorporation of203Pb to panitumumab. Reductive SDS-PAGE conditions resulted in the formation of panitumumab light and heavy chains, which were visible at 25 and 50 kDa, respectively, indicating that both antibody portions were modified with the PSC chelator and labelled with203Pb (Figure 1 D).

[0107] 203Pb-labeling and purification of radioimmunoconjugate203Pb-PSC- panitumumab provided isolated radiochemical yields of 41.5 ± 8 % (n = 5) at a molar activity of 1.2 ± 0.35 GBq / mg. The radiochemical purity of the isolated radioimmunoconjugates was greater than 99%, as analyzed by radio-TLC. Purified radioimmunoconjugates were >95% stable in human AB-type serum over 48 h.Example 5 Cellular uptake of203Pb-PSC-panitumumab

[0108] EGFR-expressing FaDu cells were incubated with203Pb-PSC- panitumumab, and cell uptake was measured at different time points.203Pb-PSC- panitumumab uptake in FaDu cells increased until it reached a plateau at 60 min (Figure 2A). EGFR specificity of cellular uptake was confirmed with blocking studies using different amounts of panitumumab (0.5 mg - 10 mg), demonstrating 70%, 85%, and 95% blocking at 0.5 mg, 1 mg, 5 mg and 10 mg, respectively (Figure 2B). The immunoreactive fraction of203Pb-PSC-panitumumab was found to be -30%, which is lower than the reported 68% for89Zr-labelled panitumumab measured in MDA-MB-468 cells (24) (Figure 2C).Example 6Use of203Pb-PSC-panitumumab for SPECT / CT imaging

[0109] A person skilled in the art may use the invention disclosed herein to perform SPECT / CT imaging in animals or humans using203Pb-PSC-panitumumab.

[0110] To perform SPECT / CT imaging in animal, one can use animal models that have xenograft transplantation of EGFR+ human cancer cells. For example, one can use a mouse model and protocol described by Niu G. et al. (Cetuximab-Based Immunotherapy and Radioimmunotherapy of Head-Neck Squamous Cell Carcinoma. Clin. Cancer Res. 2010, 16, 2095) that transplants human head and neck squamous carcinoma cell lines SCC1 and UM-SCC-22B in athymic nude mice subcutaneously to produce an animal model for studying EGFR targeted immunotherapy and radioimmunotherapy.

[0111] After establishing an animal model with human EGFR+ tumor cell grown in the animal, a person skilled in the art may administer to the animal the203Pb-PSC- panitumumab as disclosed in the present invention and use SPECT / CT to visualize the EGFR+ tumors in the animal. In certain animals,203Pb-PSC-panitumumab may be given in dosage between 5-20 MBq, preferably between 7-15 MBq. In humans,203Pb- PSC-panitumumab may be given in dosage of 50-500 MBq, preferably between 100- 400, more preferably between 200-300 MBq.203Pb-PSC-panitumumab may be prepared in saline or phosphate-buffered saline (PBS) with or without ascorbic acid, sodium acetate, gentisic acid, a chelator (e.g. DTPA or DSMA), and ethanol at concentrations that are suitable for human or animal use. The SPECT / CT imaging on animals is a well-established technique. For example, Bauer D. et al. (212Pb-Pretargeted Theranostics for Pancreatic Cancer. J Nucl Med 2024; 65:109-116) describe the use of a Mediso nanoScan SPECT / CT device equipped with a high-resolution, low-energy, multipinhole collimator detecting the203Pb characteristic x-rays between 70 and 90 keV. Prieto E. et al. (Performance evaluation of a preclinical SPECT / CT system for multianimal and multi-isotope quantitative experiments. Scientific Reports 12:18161 ; 2022) describes a SPECT / CT system and protocol for imaging animals. Kvassheim M. et al. (Imaging of212Pb in mice with a clinical SPECT / CT. EJNMMI Physics (2023) 10:47) describes imaging of mice with212Pb-PSMA targeted radiopharmaceutical using SPECT / CT.Example 7Biodistribution study of212Pb-PSC-panitumumab

[0112] A person skilled in the art can produce mouse model bearing human EGFR+ tumors as described above. Mice can be euthanized after the injection of203Pb- PSC-panitumumab disclosed herein. Tissues and organs can be collected and weighed for v-counting. The uptake was calculated as injected dose percentage per gram of each tissue (ID% / g). See Bauer D. et al.212Pb-Pretargeted Theranostics for Pancreatic Cancer. J Nucl Med 2024; 65:109-116; and Li et al. Preclinical Evaluation of a Lead Specific Chelator (PSC) Conjugated to Radiopeptides for203Pb and212Pb-Based Theranostics. Pharmaceutics 2023, 15, 414.Example 8Use of212Pb-PSC-panitumumab for treating EGFR+ tumors

[0113] A person skilled in the art may use212Pb-PSC-panitumumab for treating EGFR+ human tumors or cancers in a subject. For animal study, one can use212Pb- PSC-panitumumab disclosed herein to treat EGFR+ human tumor xenograft animals, using the animal model described above. In certain animals,212Pb-PSC-panitumumab can be administered in dose between 0.18-7.4 MBq, preferably between 0.37-3.7 MBq, more preferably between 0.74-1.85 MBq. In humans,212Pb-PSC-panitumumab can be administered in dose between 18-555 MBq, preferably between 37-370 MBq, and more preferably between 74-185 MBq.212Pb-PSC-panitumumab may be in prepared in saline or phosphate-buffered saline (PBS) with or without ascorbic acid, sodium acetate, gentisic acid, a chelator (e.g. DTPA or DSMA), and ethanol at concentrations that are suitable for human or animal use. Freeman, D.J. et al. (Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer. Mol Cancer 11 , 47 (2012)) described a protocol for treating EGFR+ tumors in mice with panitumumab. Ku A. et al. (Dose predictions for [177Lu]Lu-DOTA-panitumumab F(ab')2 in NRG mice with HNSCC patient-derived tumour xenografts based on [64Cu]Cu-DOTA-panitumumab F(ab')2 - implications for a PET theranostic strategy. EJNMMI Radiopharm Chem. 2021 Aug 12;6(1 ):25) described protocol and results of using [177Lu]Lu-DOTA-panitumumab F(ab')2 for treating mice bearing human EGFR+ tumor. A person skilled in the art can use the protocols described in these references to perform studies on using212Pb-PSC- panitumumab for treating EGFR+ tumors in animals. Panitumumab has been studied extensively in clinical trials and approved by the Food and Drug Administration for treating human cancers (e.g., Van Cutsem E, Peeters M, Siena S, et al. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol. 2007b; 25: 1658-64; www.accessdata.fda.gov / drugsatfda_docs / label / 2009 / 125147s080lbl.pdf).

[0114] Accordingly,212Pb-PSC-panitumumab can be used to target EGFR+ tumors in humans, and with the alpha emission energy from212Pb killing the tumor cells.REFERENCES1. Li M, Sagastume EA, Lee D, McAlister D, DeGraffenreid AJ, Olewine KR, et al. 203 / 212Pb Theranostic Radiopharmaceuticals for Image-guided Radionuclide Therapy for Cancer. CMC. 2020 Dec 8;27(41):7003-31 .2. Sun J, Huangfu Z, Yang J, Wang G, Hu K, Gao M, et al. Imaging-guided targeted radionuclide tumor therapy: From concept to clinical translation. Advanced Drug Delivery Reviews. 2022 Nov; 190:114538.3. Chan TG, O'Neill E, Habjan C, Cornelissen B. Combination Strategies to Improve Targeted Radionuclide Therapy. J Nucl Med. 2020 Nov;61 (11 ):1544-52.4. Kerr CP, Grudzinski JJ, Nguyen TP, Hernandez R, Weichert JP, Morris ZS. Developments in Combining Targeted Radionuclide Therapies and Immunotherapies for Cancer Treatment. Pharmaceutics. 2022 Dec 30; 15(1 ): 128.5. Gill MR, Falzone N, Du Y, Vallis KA. Targeted radionuclide therapy in combined- modality regimens. The Lancet Oncology. 2017 Jul; 18(7):e414-23.Pandit-Taskar N. Targeted Radioimmunotherapy and Theranostics with Alpha Emitters. Journal of Medical Imaging and Radiation Sciences. 2019 Dec;50(4):S41-4. Gallivanone F. Targeted radionuclide therapy frontiers in theranostics. Front Biosci. 2017;22(10):1750-9. Srivastava SC. Paving the Way to Personalized Medicine: Production of Some Promising Theragnostic Radionuclides at Brookhaven National Laboratory. Seminars in Nuclear Medicine. 2012 May;42(3): 151-63. Qaim SM, Scholten B, Neumaier B. New developments in the production of theranostic pairs of radionuclides. J Radioanal Nucl Chem. 2018 Dec;318(3): 1493-509. Srivastava SC. A Bridge not too Far: Personalized Medicine with the use of Theragnostic Radiopharmaceuticals. Journal of Postgraduate Medicine, Education and Research. 2013 Mar;47(1 ):31-46. Nelson BJB, Wilson J, Andersson JD, Wuest F. High yield cyclotron production of a novel 133 / 135La theranostic pair for nuclear medicine. Sci Rep. 2020 Dec 17;10(1 ):22203. Nelson BJB, Wilson J, Schultz MK, Andersson JD, Wuest F. High-yield cyclotron production of 203Pb using a sealed 205TI solid target. Nuclear Medicine and Biology. 2023 Jan;116-117:108314. Kokov KV, Egorova BV, German MN, Klabukov ID, Krasheninnikov ME, Larkin- Kondrov AA, et al. 212Pb: Production Approaches and Targeted Therapy Applications. Pharmaceutics. 2022 Jan 13; 14(1 ): 189. McNeil BL, Robertson AKH, Fu W, Yang H, Hoehr C, Ramogida CF, et al. Production, purification, and radiolabeling of the 203Pb / 212Pb theranostic pair. EJNMMI radiopharm chem. 2021 Dec;6(1 ):6. Mammatas LH, Verheul HMW, Hendrikse NH, Yaqub M, Lammertsma AA, Menke-van Der Houven Van Oordt CW. Molecular imaging of targeted therapies with positron emission tomography: the visualization of personalized cancer care. Cell Oncol. 2015 Feb;38(1 ):49-64.Parakh S, Lee ST, Gan HK, Scott AM. Radiolabeled Antibodies for Cancer Imaging and Therapy. Cancers. 2022 Mar 11 ; 14(6): 1454. Chamarthy MR, Williams SC, Moadel RM. Radioimmunotherapy of nonHodgkin's lymphoma: from the "magic bullets" to "radioactive magic bullets." Yale J Biol Med. 2011 Dec;84(4):391-407. Gemmete JJ, Mukherji SK. Panitumumab (Vectibix): Fig 1. AJNR Am J Neuroradiol. 2011 Jun;32(6): 1002-3. Thomas R, Weihua Z. Rethink of EGFR in Cancer With Its Kinase Independent Function on Board. Front Oncol. 2019 Aug 23;9:800. Rogers S J, Harrington KJ, Rhys-Evans P, O-Charoenrat P, Eccles SA. Biological significance of c-erbB family oncogenes in head and neck cancer. Cancer Metastasis Rev. 2005 Jan;24(1 ):47-69. Braun AC, de Mello CAL, Corassa M, Abdallah EA, Urvanegia AC, Alves VS, et al. EGFR expression in circulating tumor cells from high-grade metastatic soft tissue sarcomas. Cancer Biology & Therapy. 2018 Jun 3;19(6):454-60. Bailey LE, Ong SD. Krebs-Henseleit solution as a physiological buffer in perfused and superfused preparations. Journal of Pharmacological Methods. 1978 Aug; 1 (2): 171 -5. Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA. Determination of the immunoreactive function of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. Journal of Immunological Methods. 1984 Aug;72(1 ):77-89. Bhattacharyya S, Kurdziel K, Wei L, Riffle L, Kaur G, Hill GC, et al. Zirconium-89 labeled panitumumab: a potential immuno-PET probe for HER1 -expressing carcinomas. Nuclear Medicine and Biology. 2013 May;40(4):451-7. Dewulf J, Adhikari K, Vangestel C, Wyngaert TVD, Elvas F. Development of Antibody Immuno-PET / SPECT Radiopharmaceuticals for Imaging of Oncological Disorders — An Update. Cancers. 2020 Jul 11 ; 12(7): 1868. Fasano M, Della Corte CM, Viscardi G, Di Liello R, Paragliola F, Sparano F, et al. Head and neck cancer: the role of anti-EGFR agents in the era of immunotherapy. Ther Adv Med Oncol. 2021 Jan; 13:175883592094941 .Facca VJ, Cai Z, Ku A, Georgiou CJ, Reilly RM. Adjuvant Auger Electron- Emitting Radioimmunotherapy with [ 111 ln]ln-DOTA-Panitumumab in a Mouse Model of Local Recurrence and Metastatic Progression of Human TripleNegative Breast Cancer. Mol Pharmaceutics. 2023 Dec 4;20(12):6407-19. Aghevlian S, Lu Y, Winnik MA, Hedley DW, Reilly RM. Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111 In and 177 Lu — An EGFR-Targeted Theranostic for Pancreatic Cancer. Mol Pharmaceutics. 2018 Mar 5; 15(3): 1150-9. Ku A, Kondo M, Cai Z, Meens J, Li MR, Ailles L, et al. Dose predictions for [177Lu]Lu-DOTA-panitumumab F(ab')2 in NRG mice with HNSCC patient- derived tumour xenografts based on [64Cu]Cu-DOTA-panitumumab F(ab')2 - implications for a PET theranostic strategy. EJNMMI radiopharm chem. 2021 Dec;6(1 ):25. Ku A, Chan C, Aghevlian S, Cai Z, Cescon D, Bratman SV, et al. MicroSPECT / CT Imaging of Cell-Line and Patient-Derived EGFR-Positive Tumor Xenografts in Mice with Panitumumab Fab Modified with Hexahistidine Peptides To Enable Labeling with 99m Tc(l) Tricarbonyl Complex. Mol Pharmaceutics.2019 Aug 5;16(8):3559-68. Garmestani K, Milenic DE, Brady ED, Plascjak PS, Brechbiel MW. Purification of cyclotron-produced 203Pb for labeling Herceptin. Nuclear Medicine and Biology. 2005 Apr;32(3):301-5. Jiao R, Allen KJH, Malo ME, Yilmaz O, Wilson J, Nelson BJB, et al. A Theranostic Approach to Imaging and Treating Melanoma with 203Pb / 212Pb- Labeled Antibody Targeting Melanin. Cancers. 2023 Jul 29;15(15):3856. Miao Y, Figueroa SD, Fisher DR, Moore HA, Testa RF, Hoffman TJ, et al. 203 Pb-Labeled a-Melanocyte-Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection. J Nucl Med. 2008 May;49(5):823-9. Banerjee SR, Minn I, Kumar V, Josefsson A, Lisok A, Brummet M, et al. Preclinical Evaluation of 203 / 212 Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer. J Nucl Med.2020 Jan;61 (1 ):80-8.Ray G, Baidoo K, Wong K, Williams M, Garmestani K, Brechbiel M, et al. Preclinical evaluation of a monoclonal antibody targeting the epidermal growth factor receptor as a radioimmunodiagnostic and radioimmunotherapeutic agent. British J Pharmacology. 2009 Aug;157(8):1541-8. Nayak TK, Garmestani K, Milenic DE, Brechbiel MW. PET and MRI of Metastatic Peritoneal and Pulmonary Colorectal Cancer in Mice with Human Epidermal Growth Factor Receptor 1-Targeted 89 Zr-Labeled Panitumumab. J Nucl Med. 2012 Jan;53(1 ):113-20. Liu Z, Ma T, Liu H, Jin Z, Sun X, Zhao H, et al. 177 Lu-Labeled Antibodies for EGFR-Targeted SPECT / CT Imaging and Radioimmunotherapy in a Preclinical Head and Neck Carcinoma Model. Mol Pharmaceutics. 2014 Mar 3;11 (3):800-7. Nayak TK, Garmestani K, Baidoo KE, Milenic DE, Brechbiel MW. Preparation, Biological Evaluation, and Pharmacokinetics of the Human Anti- HER1 Monoclonal Antibody Panitumumab Labeled with 86 Y for Quantitative PET of Carcinoma. J Nucl Med. 2010 Jun;51 (6):942-50. Ho, F. K. S. (2023). Development of 197Hg-Labelled Panitumumab for Auger Electron Radioimmunotherapy of Triple-Negative Breast Cancer (Order No. 30531252). Available from ProQuest Dissertations & Theses Global. (2889518073). Chopra A. 1111n-Labeled panitumumab, a fully human monoclonal antibody directed against the extracellular domain III of the epidermal growth factor receptor. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet], Bethesda (MD): National Center for Biotechnology Information (US); 2004 [cited 2024 Jan 17], Available from: http: / / www.ncbi.nlm.nih.gov / books / NBK97967 / Sharma SK, Wuest M, Wang M, Glubrecht D, Andrais B, Lapi SE, Wuest F. Immuno-PET of epithelial ovarian cancer: harnessing the potential of CA125 for non-invasive imaging. EJNMMI Res. 2014 Dec;4(1 ):60. doi: 10.1186 / s13550- 014-0060-4. Nelson BJB, Wilson J, Andersson JD, Wuest F. Theranostic Imaging Surrogates for Targeted Alpha Therapy: Progress in Production, Purification, and Applications. Pharmaceuticals (Basel). 2023 Nov 17; 16(11 ): 1622.43. Li M. Preclinical Evaluation of a Lead Specific Chelator (PSC) Conjugated to Radiopeptides for203Pb and212Pb-Based Theranostics. Pharmaceutics 2023; 15; 414.

[0115] All documents cited in this application are hereby incorporated by reference as if recited in full herein. In the event of a conflict between the teachings of this application and those of the incorporated documents, the teachings of this application control.

[0116] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.

Claims

WHAT IS CLAIMED IS:1 . A conjugate having the formula A-(L-C)n, whereinA is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR),L is a bond or a linker,C is a lead specific chelating moiety according to Formula 5a,Formula 5a each Ri is independently hydrogen or an alkyl group,R2 is -ORi, -NFh or a bond connecting the lead specific chelating moiety, C, to the linker, L,R4 and Rs are independently hydrogen or a bond connecting the lead specific chelating moiety to the linker, L, provided (i) at least one of R4 and Rs is hydrogen, and (ii) each of R4 and Rs is hydrogen when R2 is a bond connecting the lead specific chelating moiety, C, to the linker, L, and n is at least 1 .

2. The conjugate of claim 1 wherein the conjugate further comprises a lead isotope, xPb, chelated by the lead specific chelating moiety, C.

3. The conjugate of claim 1 wherein the conjugate corresponds to Formula 1a, Formula 1b or Formula 1c:Formula 1c whereinA is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR),L is a bond or a linker,R3 is -OR1 or -NH2, wherein R1 is an alkyl group, and n is at least 1 .

4. The conjugate of claim 3 wherein the conjugate corresponds to Formula 1 a, 1 b or 1c and the conjugate further comprises a lead isotope,xPb, chelated by the lead specific chelating moiety, C.

5. The conjugate of claim 3 wherein the conjugate corresponds to Formula 1 a and the conjugate further comprises a lead isotope,xPb, chelated by the lead specific chelating moiety, C, corresponding to Formula 1a-1 :The conjugate of claim 1 , corresponding to Formula 2a:whereinA and n are as defined in claim 1 , Ri is hydrogen, andZ is -NH- and is the residue of a primary amine moiety comprised by A.

7. The conjugate of claim 6 wherein the conjugate further comprises a lead isotope, xPb, chelated by the lead specific chelating moiety, C, corresponding to Formula 2a-1 :Formula 2a- 1 whereinA and n are as defined in claim 1 , and xPb is an isotope of lead.

8. The conjugate of claim 1 , corresponding to Formula 2b:whereinA and n are as defined in claim 1 , andZ is -NH- and is the residue of a primary amine moiety comprised by A.

9. The conjugate of claim 8 wherein the conjugate further comprises a lead isotope, xPb, chelated by the lead specific chelating moiety, C, corresponding to Formula 2b-1whereinA and n are as defined in claim 1 , and xPb is an isotope of lead.

10. The conjugate of claim 1 , corresponding to Formula 2c:A and n are as defined in claim 1 , andZ is -NH- and is the residue of a primary amine moiety comprised by A.11 . The conjugate of claim 10 wherein the conjugate further comprises a lead isotope,xPb, chelated by the lead specific chelating moiety, C, corresponding to Formula 2c-1 :Formula 2c-1 whereinA and n are as defined in claim 1 , and xPb is an isotope of lead.

12. A process for the preparation of a compound suitable for radiolabeling comprising reacting a binding moiety A that is selective for epidermal growth factor receptor (EGFR), and that is any of a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein, and comprises a lysine residue, with a lead specific chelating moiety C corresponding to any of Formulas 3a, 3b and 3c:whereinRi is hydrogen.

13. A method of radiolabeling a conjugate corresponding to any of Formula 1a,Formula 1 b or Formula 1 c:Formula 1bFormula 1c whereinA is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR),L is a bond or linker,R3 is -OR1 or -NH2, wherein R1 is an alkyl group, and n is at least 1 , the method comprising: combining the conjugate with a lead radioisotope in an aqueous solution at a temperature of no more than 60°C.

14. The method according to claim 13, wherein the conjugate comprises any of Formula 2a, Formula 2b, or Formula 2c:whereinA is a binding moiety comprising a monoclonal antibody, a polyclonal antibody, an antibody fragment or a synthetic protein selective for epidermal growth factor receptor (EGFR), n is at least 1 ,Ri is hydrogen, andZ is -NH- and is the residue of a primary amine moiety comprised by A.

15. A method of imaging or diagnosing a subject suffering from a cancer associated with overexpression of epidermal growth factor receptor (EGFR), the method comprising administering the conjugate of any preceding claim, as radiolabeled with a lead radioisotope203Pb, to the subject, and imaging tissue having the radiolabeled conjugate compound bound thereto.

16. A method of treating a subject suffering from a cancer associated with overexpression epidermal growth factor receptor (EGFR), the method comprisingadministering the conjugate of any preceding claim, as radiolabeled with a lead radioisotope212Pb, to the subject, in a dosage sufficient to kill tumor cells.

17. A method of diagnosing and treating a subject suffering from a cancer associated with overexpression of EGFR, the method comprising: diagnosing the subject by administering the conjugate of any preceding claim to the subject, as radiolabeled with a lead radioisotope where203Pb, and imaging tissue having the radiolabeled conjugate compound bound thereto to diagnose the subject as being afflicted with the cancer, and treating the subject diagnosed as being afflicted by the cancer, by administering the conjugate of any preceding claim to the subject, as radiolabeled with a lead radioisotope212Pb, in a dosage sufficient to kill cancer cells.

18. The use of the conjugate according to any preceding claim for medical imaging and / or diagnosis of a subject suffering from cancer, as radiolabeled with203Pb.

19. The use of the conjugate according to any preceding claim for treatment of a subject suffering from cancer, as radiolabeled with212Pb.

20. An aqueous formulation comprising the conjugate of any preceding claim and as radiolabeled with a lead radioisotope, wherein the formulation comprises a buffer and optionally further comprises any one or more of ascorbic acid, sodium acetate, gentisic acid, DTPA, DSMA, and ethanol.21 . The aqueous formulation according to claim 20, wherein the lead radioisotope is203Pb (Pb-203), and is provided in the formulation in an amount sufficient to provide a dosage of 50-500 MBq, 100-400 MBq, and / or 200-300 MBq.

22. The aqueous formulation according to claim 20, wherein the lead radioisotope is212Pb (Pb-212), and is provided in the formulation in an amount sufficient to provide a dosage of 18-555 MBq, 37-370 MBq, and / or 74-185 MBq.

23. The conjugate and / or method according to any of claims 1-2, wherein the lead isotope is selected from the group consisting of Pb-196, Pb-197, Pb-198, Pb-199, Pb- 200, Pb-201 , Pb-202, Pb-203, Pb-205, Pb-209, Pb-210, Pb- 211 , Pb-212, Pb-213, Pb- 214, Pb-215, and Pb-216.

24. The conjugate and / or method according to any of claims 1-2, wherein the lead radioisotope is203Pb (Pb-203) or212Pb (Pb-212).

25. The conjugate and / or method according to any preceding claim, wherein the binding moiety A is an antibody selected from the group consisting of cetuximab, panitumumab and necitumumab.

26. The conjugate and / or method according to claim 25, wherein the binding moiety A is panitumumab.

27. The conjugate and / or method according to any preceding claim wherein n at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 and / or at least 10.

28. The conjugate and / or method according to any preceding claim, wherein the binding moiety A is an antibody, and wherein the binding moiety A is bound to leadspecific chelating moiety on both the light and heavy chains.

29. The method of radiolabeling according to claim 13 or 14, wherein the conjugate is combined with the lead radioisotope at a temperature of no more than 55°C, no more than 50°C, no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C, and / or no more than 20°C.

30. The method of radiolabeling according to any one of claims 13-14 and 29, wherein the conjugate is combined with the lead radioisotope at a temperature that is in the range of from 20°C to 60°C, 20°C to 40°C, 20°C to 30°C, and / or about 25°C.31 . The method of radiolabeling according to any one of claims 13-14 and 29-30, wherein the conjugate is combined with the lead radioisotope for no more than 30minutes, no more than 20 mins, no more than 15 mins, and / or no more than 10 mins, and at least 1 min, at least 3 mins, at least 4 mins, and / or at least 5 mins.

32. The method of radiolabeling according to any one of claims 13-14 and 29-31 , wherein the conjugate is combined with the lead radioisotope in an aqueous solution having a pH in a range of from 4 to 6, and / or a pH of about 5.

33. The method of imaging or diagnosing the cancer according to claim 15, wherein the tissue is imaged via single-photon emission computed tomography (SPECT) imaging, optionally in combination with computed tomography (CT) imaging.

34. The method according to any one of claims 15-17 and 33, wherein the cancer that is associated with overexpression of EGFR is any of colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, and breast cancer.

Images