TSHR-targeted radiopharmaceuticals for diagnosis and therapy of thyroid cancer

Abstract

Provided herein are thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceuticals, comprising a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide (e.g., 89Zr-DFO-TSHR-Ab or 64Cu-NOTA-TSHR-Ab). Also provided are methods of making the TSHR-targeting radiopharmaceuticals, and their use in the diagnosis and treatment of diseases (e.g., cancer).

Description

Docket No. 31134 / 2023-456 / PCTSHR-TARGETED RADIOPHARMACEUTICALS FOR DIAGNOSIS AND THERAPY OF THYROID CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 682,927 filed on August 14, 2024, the disclosures of which are fully incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

[0002] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 2023-456_SeqListing.xml; Size: 3,686 bytes; Created: August 14, 2025), which is incorporated by reference in its entirety.FIELD

[0003] The present disclosure in the field of biochemistry, immunology, and medicine relates to antibodies (“Abs”) and other proteins or bioactive molecules conjugated to imaging or therapeutic radionuclides (e.g., radioactive metals) for the diagnosis or treatment of disease.BACKGROUND

[0004] Thyroid cancer is the most common endocrine cancer and its incidence is rising in the United States and globally [1], Thyroid cancer patients have widely different clinical outcomes depending on the pathological subtype. Advanced thyroid cancers, such as poorly differentiated thyroid cancer, anaplastic thyroid cancer, and metastatic thyroid cancers such as papillary, follicular, and oncocytic (OTC, also called Hurthle cell) thyroid cancers, are aggressive and hard to treat, and are often refractory to radiation ablation with iodine-131 (1-131) and other standard therapies [2, 3],

[0005] Thyroid-stimulating hormone (TSH), via its receptor (TSHR), increases thyroid hormone levels by upregulating expression of the sodium iodide symporter (NIS), thyroid peroxidase, and thyroglobulin genes, all required for efficient iodide incorporation in thyroid hormones [4, 5], TSHR, also called Thyrotropin receptor, is a surface glycoprotein receptor and serves as a key regulator of thyroid function and growth. TSHR is a thyroid selective receptor that is predominantly expressed on the basolateral membrane of normal thyroid follicular cells (estimated 5000 receptors per cell) [6], As a member of the G protein-coupled receptor family, TSHR is also abundantly expressed on the surface of thyroid tumor cells [6, 7], and is internalized into the cells after binding to TSH or other ligands [8], Given its unique expression, TSHR has the potential to serve as a compelling target for radiopharmaceutical development for the imaging and therapy of thyroid cancer [6], In vivo quantification of TSHR expression and noninvasive monitoring of the ligand and tumor engagement are critical to advancing the successful development of emerging precision TSHR-targeted companion diagnostics and therapies.

[0006] Positron emission tomography (PET) allows for noninvasive characterization and quantification of a biological target and there are several radiopharmaceuticals including 2-[18F]fluoro-2-deoxy-D-glucose (FDG), [124l]lodine, [18F]tetrafluoroborate that have been used in PET imaging of thyroid cancer by targeting glucose metabolism or human NIS expression [9-11], However, these radiotracers are not directly reflective of TSHRDocket No. 31134 / 2023-456 / PC expression, and as a group fail to adequately identify and provide prognostic guidance on TSHR expressing thyroid carcinomas. TSH (Kd = 50 nM) is the endogenous ligand targeting TSHR; however, radioiodinated recombinant human TSH (rhTSH, Thyrogen) has shown low tumor uptake and low tumor / background (T / B) ratio in part due to low binding affinity

[0012] , While a few small molecules and antibodies have been developed for TSHR targeted therapy [13, 14], due to limited binding affinities of these reported agents, only two rhTSH analog antibodies (TR1401 and TR1402) have been labeled with Tc-99m, for SPECT imaging and have undergone preclinical evaluation [15, 16], More recently, a TSHR agonist, TR1402, has been radiolabeled with Zr-89 for PET imaging of TSHR to validate TSHR as a target for imaging differentiated thyroid cancer and undergone in-vitro and in vivo analysis

[0017] ,

[0007] Differentiated thyroid cancer (DTC) accounts for about 95% of all thyroid carcinomas. Radioiodine (RAI), the first radiotheranostic, has been used to diagnose and treat DTC since the 1940s and remains the most prescribed treatment to specifically and systemically eradicate tumor cells by ionizing radiation. RAI is transported through the sodium / iodide symporter (NIS) into thyrocytes, and subsequently is oxidated and covalently bound to thyroglobulin. However, despite the wide application of RAI, 5-15% of DTC and more 50% of metastatic DTCs are unresponsive to RAI, resulting in poor outcomes (-10% a 10-year survival). With the current renaissance of clinical radiotheranostics, targeted radiopharmaceutical therapy (TRT) utilizes the integration of modern cancer biology, pharmaceutical sciences, and radiochemistry to identify novel biological targets and synthesize unique radiopharmaceuticals to deliver deadly DNA damaging ionizing radiation directly to tumor cells, which may overcome RAI resistance on DTC. Compared to traditional RAI therapy with p-emitter 1-131, a- emitters deliver a higher amount of energy over just a few cell diameters for potent, precise, and efficiently targeted cell kill and minimized toxicity to non-targeted surrounding cells. New generation of targeted p-emitter radiopharmaceutical therapy or targeted a-emitting radiopharmaceutical therapy could be a promising treatment modality to overcome RAI resistance on thyroid cancer.

[0008] A need therefore exists for new TSHR-targeted radiopharmaceuticals having improved properties, such as increased specificity and affinity compared to known radiopharmaceuticals that target TSHR for thyroid cancer diagnosis and therapy.SUMMARY

[0009] Disclosed herein are thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceuticals, comprising a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide. In some embodiments, the TSHR-targeting moiety is an antibody, e.g., K1-70. In some embodiments, the chelator moiety comprises desferrioxamine (DFO) or 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA). In some embodiments, the radionuclide is89Zr or64Cu. In some embodiments, provided are particular TSHR-targeting radiopharmaceuticals including89Zr-DFO-TSHR-Ab and64Cu-NOTA-TSHR-Ab, wherein Ab is an antibody (e.g., K1-70).Docket No. 31134 / 2023-456 / PC

[0010] Further disclosed are methods of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a therapeutically-effective amount of a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide. In various embodiments, the thyroid-stimulating hormone receptor- (TSHR-) targeting radiopharmaceutical is a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical disclosed herein, such as89Zr-DFO-TSHR-Ab and64Cu-NOTA-TSHR-Ab, wherein Ab is an antibody (e.g., K1-70). In various embodiments, the disease or disorder is cancer, such as thyroid cancer. In various embodiments, the patient has previously been administered anti-TSHR targeted therapy, e.g., TSHR- targeted chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

[0011] Further disclosed are methods of diagnosing a disease or disorder in a patient, comprising the steps of: (a) administering to the patient a thyroid-stimulating hormone receptor- (TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide; (b) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours 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 2:1 or greater. In various embodiments, the thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical is a thyroid- stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical disclosed herein, such as89Zr-DFO-TSHR- Ab and64Cu-NOTA-TSHR-Ab, wherein Ab is an antibody (e.g., K1-70). In various embodiments, the disease or disorder is cancer, such as thyroid cancer. In various embodiments, the patient has previously been administered anti-TSHR targeted therapy, e.g., TSHR-targeted chimeric antigen receptor T-cell therapy (CAR T- cell therapy).

[0012] Further disclosed are methods of treating a disease or disorder in a patient, comprising the steps of: (a) diagnosing a disease or disorder in a patient, comprising i) administering to the patient a thyroid-stimulating hormone receptor- (TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide; (ii) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and (iii) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater; and b) treating the disease or disorder by administering to the patient a therapeutically-effective amount of the thyroid-stimulating hormone receptor- (TSHR-) targeting radiopharmaceutical. In various embodiments, the thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical is a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical disclosed herein, such as89Zr-DFO-TSHR-Ab,64Cu-NOTA-TSHR-Ab,177Lu-DOTA-TSHR-Ab,67Cu-NOTA-TSHR-Ab, or225Ac-DOTA-TSHR-Ab, wherein Ab is an antibody (e.g., K1-70). In various embodiments, the disease or disorderDocket No. 31134 / 2023-456 / PC is cancer, such as thyroid cancer. In various embodiments, the patient has previously been administered anti- TSHR targeted therapy, e.g., TSHR-targeted chimeric antigen receptor T-cell therapy (CAR T-cell therapy).BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A-1B Synthesis and characterization of89Zr-DFO-TSHR-Ab (Construct A1). (FIG. 1A) Conjugation of chelator DFO-Bn-NCS to human antibody TSHR-Ab K1-70 in basic aqueous conditions and89Zr- radiolabeling of DFO-TSHR-Ab. (FIG. 1B) After purification of reaction mixture using a PD-10 desalting column, quality control of89Zr-DFO-TSHR-Ab was performed using radio-TLC.

[0014] FIG. 2A-2D In vitro evaluation of89Zr-DFO-TSHR-Ab demonstrated specificity and high affinity binding to TSHR expressing cells. (FIG. 2A) IHC of TSHR expression in WT and TSHR-expressing THJ-529T, FTC-133, and K562 demonstrated strong TSHR expression in TSHR-transduced cells (brown staining) compared to their respective WT cells. (FIG. 2B)89Zr-DFO-TSHR-Ab showed substantially higher uptake in all TSHR-expressing K562, THJ-529T, and FTC-133 cell lines than their WT cells, and their uptake in TSHR-positive tumor cells could be blocked with TSHR antibody K-70 (20 pg). (FIG. 2C) The binding affinity of89Zr-DFO-TSHR-Ab on TSHR- K562 cells using a saturation binding assay demonstrated a Kd value of 0.58 nM. (FIG. 2D) A competition radioligand binding assay using89Zr-DFO-TSHR-Ab, demonstrated an IC50 value for TSHR antibody of 0.81 nM on TSHR-K562 cells.

[0015] FIG. 3A-3D, Zr-89 labeled TSHR antibody detects TSHR-expressing tumors. (FIG. 3A) Coronal projection images of PET-CT after the injection of 50 piCi of89Zr-DFO-TSHR-Ab in a NSG mouse with implanted WT K652 tumor in the left flank (right-facing arrows) and implanted TSHR expressing tumor TSHR-K652 in the right flank (left-facing arrows). The fused PET / CT images at 24, 48, and 72 hours demonstrated higher89Zr-DFO- TSHR-Ab uptake in the TSHR-expressing tumor compared to the W.T. tumor. (FIG. 3B) Quantitative uptake of89Zr-DFO-TSHR-Ab in tumor and various tissues (liver, kidney, muscle, and blood) at 24, 48, and 72 hours. (FIG. 3C) Coronal projection images of PET-CT after the coinjection of 50 piCi of89Zr-DFO-TSHR-Ab with 100 pig unlabeled TSHR-Ab in an NSG mouse with implanted WT K652 tumor in the left flank (right-facing arrows) and implanted TSHR expressing tumor TSHR- K652 in the right flank (left-facing arrows) are shown. The fused PET / CT images at 24, 48, and 72 hours demonstrated higher89Zr-DFO-TSHR-Ab uptake in the TSHR- expressing tumor compared to the WT tumor. (FIG. 3D) Quantitative uptake of89Zr-DFO-TSHR-Ab coinjection with TSHR antibody in tumor and various tissues (liver, kidney, muscle, and blood) at 24, 48, and 72 hours are shown.

[0016] FIG. 4A-4C Comparison of PET imaging of TSHR with I without TSHR antibody coinjection. (FIG. 4A) Time-activity curves of all tumors after injecting89Zr-DFO-TSHR-Ab with or without TSHR antibody coinjection are shown. (FIG. 4B) Tumor-to-nontarget organs (liver, muscle, and blood) ratios of89Zr-DFO-TSHR-Ab without coinjection of TSHR antibody from 24 to 72 hours are shown. (FIG. 4C) Tumor-to-nontarget organs (liver, muscle, and blood) ratios of89Zr-DFO-TSHR-Ab with coinjection of TSHR antibody from 24 to 72 hours are shown.Docket No. 31134 / 2023-456 / PC

[0017] FIG. 5A-5C Ex vivo evaluation of89Zr-DFO-TSHR-Ab showed enhanced uptake with TSHR antibody coinjection. (FIG. 5A) The radiotracer biodistribution at 72 h post-injection (n = 3) is shown. (FIG. 5B) Tumor-to- nontarget organs (K562 WT, liver, kidney, muscle, and blood) ratios of89Zr-DFO-TSHR-Ab with or without TSHR antibody coinjection are shown. (FIG. 5C) Immunohistochemical staining of TSHR-K562 and K562 WT tumors is shown.

[0018] FIG. 6A-6F In vitro evaluation of89Zr-DFO-TSHR-Ab in THJ-529T and FTC-133 cell lines. (FIG. 6A) and (FIG. 6D)89Zr-DFO-TSHR-Ab uptake in TSHR-expressing cell line TSHR-THJ-529T, TSHR-FTC-133 and their wild type THJ-529T, FTC-133, in presence or absence of TSHR antibody (20 pig) respectively. (FIG. 6B) and (FIG. 6E) Binding affinity of89Zr-DFO-TSHR-Ab on TSHR-THJ-529T and TSHR-FTC-133 cells, respectively ( Kd= 2.31 nM for TSHR-THJ-529T and Kd= 3.67 nM for TSHR-FTC-133). (FIG. 6C) and (FIG. 6F) Competition radioligand binding assay using89Zr-DFO-TSHR-Ab, demonstrating IC50 value for TSHR antibody 1 .04 nM on TSHR-THJ-529T cells, and 0.84 nM on TSHR-FTC133 cells.

[0019] FIG. 7A-7I Zr-89 radiolabeled TSHR antibody detects TSHR expressing tumor in absence or presence of antibody. (FIG. 7A-I) Coronal projection images of89Zr-DFO-TSHR-Ab PET-CT after injection of 50 piCi of89Zr-DFO-TSHR-Ab in a NSG mouse with implanted wild type (WT) K652 tumor in the left flank (right-facing arrows) and implanted TSHR expressing tumor TSHR+ K652 in the right flank (left-facing arrows). 24 hr fused (FIG. 7A) CT (FIG. 7B) and PET (FIG. 7C) images demonstrate increased89Zr-DFO-TSHR-Ab uptake in the TSHR transfect tumor (SUVmax 1 .6) compared to the WT tumor (SUVmax 0.8). The 48 hr fused (FIG. 7D) CT (FIG. 7E) and PET (FIG. 7F) images and 72 hr fused (FIG. 7G) CT (FIG. 7H) and PET (I) images show persistent uptake in TSHR-K652 tumor (SUVmax 1.3 and 1.2 respectively) and unchanged mild uptake in the K652 WT tumor (SUVmax 0.7 and 0.6 respectively).89Zr-DFO-TSHR-Ab PET otherwise demonstrates expected antibody distribution including uptake in the liver, spleen, and bone marrow. (FIG. 7A’-I’) Coronal projection images of89Zr-DFO-TSHR-Ab PET-CT after co-injection of 50 pjCi of89Zr-DFO-TSHR-Ab with 100 g unlabeled TSHR-Ab in a NSG mouse with implanted wild type K652 tumor in the left flank (right-facing arrows) and implanted TSHR expressing tumor TSHR-K652 in the right flank (left-facing arrows). The 24 hr fused (FIG. 7 A’) CT (FIG. 7B’) and PET (FIG. 7C’) images demonstrate intense89Zr-DFO-TSHR-Ab uptake in the TSHR transfect tumor (SUVmax 2.8) compared to the WT tumor (SUVmax 1.3). The 48 hr fused (FIG. 7D’) CT (FIG. 7E’) and PET (FIG. 7F’) images and 72 hr fused (FIG. 7G’) CT (FIG. 7H’) and PET (FIG. 7I’) images show interval increased uptake in TSHR-K652 tumor (SUVmax 2.7 and 2.9 respectively) and unchanged mild uptake in the K652 WT tumor (SUVmax 1.1 and 1.2 respectively).89Zr-DFO-TSHR-Ab PET otherwise demonstrates expected antibody distribution including uptake in the liver, spleen, and bone marrow, with decreased uptake in the liver and bone compared to the89Zr-DFO-TSHR-Ab PET-CT without co- unlabeled TSHR antibody K1-70 coinjection.

[0020] FIG. 8A-8C Representative images from the tissue microarray (TMA) slides with patient samples including immunohistochemical (IHC) staining of TSHR expression in normal thyroid (FIG. 8A) and oncocytic thyroid carcinoma XTC-UC1 tumor (FIG. 8B). FIG. 8C depicts a graph of the TSHR expression level quantifiedDocket No. 31134 / 2023-456 / PC using H-Score (0-3 intensity scoring) and the percentage of total areas corresponding to each score.**** p <0.0001, unpaired f-test.

[0021] FIG. 9A-9B89Zr-DFO-TSHR-Ab PET detects TSHR-positive OTC tumor in mice. Representative coronal images of OTC XTC-UC1 xenograft acquired 24, 72, and 120 hours after the tracer IV injection before and after TSHR-CART therapy. (FIG. 9A) The fused PET / CT images at 24, 72, and 120 hours demonstrated higher radiotracer uptake in TSHR-expressing OTC tumor before TSHR-CART therapy.89Zr-DFO-TSHR-Ab PET otherwise demonstrates expected antibody distribution including uptake in the liver, kidney, and muscle. (FIG. 9B) The fused PET / CT images at 24, 72, and 120 hours demonstrated lower radiotracer of OTC tumor after TSHR CAR-T therapy and inhibits OTC growth. Right-pointing arrow indicates rechallenged tumor xenograft after TSHR CAR-T therapy.

[0022] FIG. 9C depicts representative coronal PET / CT images of XTC.UC 1 tumor-bearing mice at 24, 72, and 120 hours post injection of89Zr-DFO-TSHR-Ab. Images show radiotracer uptake in tumors before therapy (top row, arrows), after TSHR CAR-T Therapy (middle row, arrows), and after tumor rechallenge in the same mice (bottom row, arrows).

[0023] FIG. 9D depicts a graph of the quantification of tumor uptake based on maximum standardized uptake value (SUVmax) at 24, 72, and 120 h p.i. across the three conditions (Pre-CAR-T (circle), Post-CAR-T (square), Rechallenge (triangle)).

[0024] FIG. 9E-9F depict graphs of the ratio of SUVmax of tumor to muscle (FIG. 9E) or of tumor to liver (FIG. 9F) at 24, 72, and 120 hours h p.i. across the three conditions (Pre-CAR-T (circle), Post-CAR-T (square), Rechallenge (triangle)).

[0025] FIG. 10A-10B Ex vivo studies of89Zr-DFO-TSHR-Ab in OTC tumor. (FIG. 10A) Biodistribution analysis of89Zr-DFO-TSHR-Ab in XTC.UC1 OTC tumor xenografts, obtained 24 hours and 120 hours following radiotracer administration, demonstrated high tumor retention in mice bearing XTC.UC1 OTC tumor. (FIG. 10B) TSHR IHC of XTC.UC1 tissues showed high expression.

[0026] FIG. 11 A-11 B Synthesis and characterization of64Cu-NOTA-TSHR-Ab (Construct A2). (FIG. 11 A) Conjugation of chelator NOTA-Bn-NCS to humanized antibody TSHR-Ab K1-70 in basic aqueous conditions and64Cu-radiolabeling of NOTA-TSHR-Ab. (FIG. 11 B) After purification of reaction mixture using a PD-10 desalting column, quality control of64Cu-NOTA-TSHR-Ab was performed using radio-TLC, showing 97.07% radiochemical purity of64Cu-NOTA-TSHR-Ab.

[0027] FIGs. 11C-11 F After purification of reaction mixture using a PD-10 desalting column, quality control of64Cu-NOTA-TSHR-Ab was performed using radio-HPLC and radio-TLC. (FIG. 11C) Representative HPLC of conjugate NOTA-TSHR-Ab (retention time: 4.487 min for UV at 280 nm, purity >95%,. (FIG. 11 D) Representative radio-HPLC of64Cu-NOTA-TSHR-Ab (retention time: 4.485 min for UV at 280 nm and 4.789 min for radiopeak.Docket No. 31134 / 2023-456 / PC(FIG. 11 E) Radio-TLC also showed high radiochemical purity of64Cu-NOTA-TSHR-Ab. (FIG. 11 F) In vitro stability of64Cu-NOTA-TSHR-Ab in normal saline and FBS at 1 , 4, 24, and 48 h.

[0028] FIG. 12A-12E Cell uptake, specificity, and binding affinity of [64Cu]Cu-NOTA-TSHR-Ab toTH J529TreHR+cells. (FIG. 12A) High uptake of [64Cu]Cu-NOTA-TSHR-Ab in TH J529T7SHR+cells, with specificity demonstrated by blocking with an excess of non-radioactive TSHR-Ab. *** P < 0.001 , **** P < 0.0001. (FIG. 12B) Saturation binding assay of [64Cu]Cu-NOTA-TSHR-Ab on TH J529T7SHR+cells with a Kd of 4.74 nM. (FIG. 12C) Competition binding assay of [64Cu]Cu-NOTA-TSHR-Ab on TH J529T7SHR+cells with a Ki of 0.92 nM. (FIG. 12D) uptake in THJ529TTSHR+ cells increased with incubation time extension, which become 34.53 ± 0.78 %uptake / mg at 1 h and reached 86.70 ± 4.37 %uptake / mg after a 3-h incubation. (FIG. 12E) Immunoreactivity assay of [64Cu]Cu-NOTA-TSHR-Ab was also conducted which exhibited high immunoreactivity with a r value of 0.87.

[0029] FIG. 13A-13B Cu-64 labeled TSHR antibody K1-70 detects TSHR-expressing tumors. (FIG. 13A) Coronal projection images of PET-CT after the injection of 150 piCi of64Cu-NOTA-TSHR-Ab in a NSG mouse with implanted DTC tumor THJ-529T WT in the left flank (right-facing arrow) and implanted TSHR expressing DTC tumor THJ-529T-TSHR in the right flank (left-facing arrow). The fused PET / CT images at 4, 18 24, and 48 hours demonstrated higher64Cu-NOTA-TSHR-Ab uptake (SUV: 20-30) and prolonged retention (> 48 h) in the TSHR-expressing tumor compared to the W.T. tumor. (FIG. 13B) Quantitative uptake of64Cu-NOTA-TSHR-Ab in tumor at 4, 18 24, and 48 hours.

[0030] FIG. 13C-13F [64Cu]Cu-NOTA-TSHR-Ab PET / CT images and TSHR expression in the subcutaneous thyroid tumor mouse model. (FIG. 13C) Representative PET / CT images of TH J529T7SHR+41447' tumor-bearing mice at 1 , 2, 4, 18, 24, and 48 h p.i. left arrow: THJ529T7SHR+tumor, right arrow: TH J529T1447tumor. (FIG. 13D) SUVmax values of tumor7SHR+, tumor1447', liver, blood, bone, kidney, and muscle from PET images. * P < 0.05, *** P < 0.001, **** P < 0.0001. (FIG. 13E) T / NT ratio of tumor7SHR+to tumor1447', liver, blood, bone, kidney, and muscle. (FIG. 13F) Representative immunohistochemistry images of tumor tissues showed strong TSHR expression in tumorTSHR+(dark staining) compared to tumor1447.

[0031] FIG. 14A-14C [64Cu]Cu-NOTA-TSHR-Ab PET / CT images with different doses of non-radioactive TSHR-Ab coinjection. (FIG. 14A) Representative PET / CT images of TH J529T7SHR+tumor-bearing mice at 18 h post co-injection with 100, 50, 25, 0 pig of non-radioactive TSHR-Ab, respectively. Left arrow: THJ529TTSHR+tumor, right arrow: spleen. (FIG. 14B) SUVmax values of tumor, liver, blood, bone, kidney, and muscle from PET images. *** P < 0.001 , **** P < 0.0001. (FIG. 13C) T / NT ratio of tumor to liver, blood, bone, kidney, and muscle. * P < 0.05, *** P < 0.001, **** P < 0.0001.

[0032] FIG. 15A-15B (FIG. 15A) Biodistribution of [64Cu]Cu-NOTA-TSHR-Ab co-injection with 100 pig nonradioactive TSHR-Ab in mice with both TH J529T1447’ tumor (left flank) and TH J529T7SHR+tumor (right flank) at 18 and 48 h p.i. *** P < 0.001 , **** P < 0.0001. (FIG. 15B) Biodistribution of [64Cu]Cu-NOTA-TSHR-Ab with 100, 50,Docket No. 31134 / 2023-456 / PC25, or 0 g non-radioactive TSHR-Ab co-injection in mice with TH J529TreHR+tumor at 18 h p.i. * P < 0.05, ** P < 0.01, 0.0001.

[0033] FIG. 16A-16E Comparison of [64Cu]Cu-NOTA-TSHR-Ab and [18F]FDG PET / CT imaging in advanced thyroid cancer mouse model. (FIG. 16A) Representative [18F]FDG PET / CT image of THJ529TreHR+tumor-bearing mouse at 30 min p.i. left arrow: TH J529TreHR+tumor. (FIG. 16B) Representative [64Cu]Cu-NOTA-TSHR-Ab PET / CT image of THJ529TreHR+tumor-bearing mouse at 18 h p.i. left arrow: TH J529TreHR+tumor. (FIG. 16C) Quantitative analysis of tumor from PET images. **** P < 0.0001. (FIG. 16D) Comparison of the tumor to muscle ratio between [18F]FDG and [64Cu]Cu-NOTA-TSHR-Ab PET images. ** P< 0.01. (FIG. 16E) Quantitative analysis of blood, bone, kidney, liver, muscle, and spleen from PET images. ** P < 0.01, *** P < 0.001.

[0034] FIG.17 Synthesis of64Cu-NOTA-TSHR-Fab (Construct A65) or64Cu-NOTA-TSHR-ScFv (Construct A66). The bifunctional chelator p-SCN-Bn-NOTA was conjugated to the antibody fragments Fab or scFv under basic aqueous conditions (pH 8-9) at 37 °C for 2 hours and64Cu-labeling of NOTA-TSHR-Fab / scFv was performed under acidic aqueous condition (pH 5-6) at 37 °C for 1 hour.

[0035] FIG.18A’-18C” Characterization of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv and in vitro stability. Representative radio-TLC chromograph of64Cu-NOTA-TSHR-Fab (FIG.18A’) and64Cu-NOTA-TSHR- scFv (FIG.18A”). Representative HPLC chromograph of64Cu-NOTA-TSHR-Fab (FIG.18B’) and64Cu-NOTA- TSHR-scFv (FIG.18B”) at UV of 280 nm. (C) In vitro stability of64Cu-NOTA-TSHR-Fab (FIG.18C’) and64Cu- NOTA-TSHR-scFv (FIG.18C”) in 10% FBS and normal saline at 0, 1, 4, 24, 48, and 72 h.

[0036] FIG.19A’-190” Cell uptake, specificity, and binding affinity of64Cu-NOTA-TSHR-Fab or64Cu-NOTA- TSHR-ScFv. FIG.19A’-19A” High uptake of64Cu-NOTA-TSHR-Fab, (Fig.19A’)64Cu-NOTA-TSHR-scFv (FIG.19A”) and in THJ529TTSHR+cells, with specificity demonstrated by blocking with an excess of nonradioactive TSHR-Ab. **** P < 0.0001. FIG.19B’-19B” Saturation binding assay of64Cu-NOTA-TSHR-Fab (FIG.19B’)64Cu-NOTA-TSHR-scFv (FIG.19B”) on THJ529TTSHR+cells with the Kd of 13.02 nM and 4.57 nM.FIG.19C’-19C” Competition binding assay of64Cu-NOTA-TSHR-Fab (Fig.19C’)64Cu-NOTA-TSHR-scFv (FIG.19C”) on THJ529TTSHR+cells with the Ki of 4.85 nM and 1 .84 nM.

[0037] FIG. 20A-20D64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv PET / CT images in the subcutaneous thyroid tumor mouse model. FIG. 20A depicts representative PET / CT images of THJ529TTSHR+tumor-bearing mice at 1, 4, 18, and 24 h post-injection of 64Cu-NOTA-TSHR-Fab with or without nonradioactive TSHR-Ab. Red arrow: THJ529TTSHR+tumor. FIG. 20B depicts representative PET / CT images of THJ529TTSHR+tumor-bearing mice at 1, 4, 18, and 24 h post-injection of64Cu-NOTA-TSHR-scFv with or without nonradioactive TSHR-Ab.Yellow arrow: THJ529TTSHR+tumor. FIG. 20C depicts SUVmax values of tumorTSHR+post-injection of64Cu-NOTA- TSHR-Fab from PET images. **** P < 0.0001 . FIG. 20D depicts SUVmax values of tumorTSHR+post-injection of64Cu-NOTA-TSHR-scFv from PET images. * P < 0.05, ** P < 0.01.

[0038] FIG. 21 A-21 B Ex vivo biodistribution of64Cu-NOTA-TSHR-Fab in mice bearing TH J529TTSHR+tumor with or without blocking at 24 hours post-injection (FIG. 21 A). ** P < 0.01 , **** P < 0.0001 . Ex vivo biodistributionDocket No. 31134 / 2023-456 / PC of 64Cu-NOTA-TSHR-scFv in mice bearing THJ529TTSHR+ tumor with or without blocking at 24 hours postinjection (FIG. 21 B). ** P < 0.01, ns > 0.05.

[0039] FIG. 22A-22B Radiosynthesis scheme of67Cu-NOTA-TSHR-Ab and its radio-TLC analysis. Anti-TSHR antibody K1-70 was conjugated with NOTA and then labeled with67Cu (FIG. 22A). FIG. 22B depicts representative radio-TLC chromograph of67Cu-NOTA-TSHR-Ab (radiochemical purity > 99%).

[0040] FIG. 23A-23C Therapeutic efficacy of67Cu-NOTA-TSHR-Ab (0.1, 0.2, and 0.4 mCi, with 25 pig for TSHR-antibody K1-70, n= 5) in comparison to the control groups (normal saline or 25 pig for TSHR-antibody K1- 70). FIG. 23A depicts average tumor growth for the mice groups treated with67Cu-NOTA-TSHR-Ab in comparison to the control groups. FIG. 23B depicts average body weights for mice group treated with67Cu- NOTA-TSHR-Ab in comparison to the control groups. FIG. 23C depicts survival data for mice treated with various doses of67Cu-NOTA-TSHR-Ab in comparison to the control groups.

[0041] Fig.24A-24B Radiosynthesis scheme of177Lu-DOTA-TSHR-Ab (Construct A17) and its radio-TLC analysis. Anti-TSHR antibody K1-70 was conjugated with DOTA and then labeled with177Lu (Fig.24A). FIG. 24B depicts representative radio-TLC chromograph of177Cu-NOTA-TSHR-Ab (radiochemical purity > 99%).

[0042] FIG. 25A-25C Therapeutic efficacy of177Lu-DOTA-TSHR-Ab (0.1, 0.2, and 0.3 mCi, with 25 pig for TSHR-antibody K1-70, n= 5) in comparison to the control groups (normal saline or 25 pig for TSHR-antibody K1- 70). FIG. 25A depicts average tumor growth for the mice groups treated with177Lu-DOTA-TSHR-Ab in comparison to the control groups. FIG. 25B depicts average body weights for mice group treated with177Lu- DOTA-TSHR-Ab in comparison to the control groups. FIG. 25C depicts survival data for mice treated with177Lu- DOTA-TSHR-Ab in comparison to the control groups.

[0043] FIG. 26A-26B Radiosynthesis scheme of225Ac-DOTA-TSHR-Ab (Construct A27) and its Radio-TLC analysis. Anti-TSHR antibody K1-70 was conjugated with DOTA and then labeled with225Ac (FIG. 26A). FIG. 26B depicts representative radio-TLC chromograph of225Ac-DOTA-TSHR-Ab (radiochemical purity > 96%).

[0044] FIG. 27A-27C Therapeutic efficacy of225Ac-DOTA-TSHR-Ab (5, 10, and 20 kBq, with 25 pig for TSHR- antibody K1-70, n= 5) in comparison to the control group (normal saline). FIG. 27A depicts average tumor growth for the mice groups treated with225Ac-DOTA-TSHR-Ab in comparison to the control group. FIG. 27B depicts average body weights for mice group treated with225Ac-DOTA-TSHR-Ab in comparison to the control group. FIG. 27C depicts survival data for mice treated with225Ac-DOTA-TSHR-Ab in comparison to the control group.DETAILED DESCRIPTION

[0045] TSHR plays an essential role in regulating the thyroid hormone and function and is a major thyroid autoantigen, which has been established as a tissue-specific target for therapeutic drug development in thyroid diseases, including hyperthyroidism known as Graves' disease, and thyroid cancer [6], Since differentiated thyroid cancer retains dependence on TSH signaling for growth, TSHR-targeted TSH suppression therapy is a standard of care to enhance radioactive iodine (RAI) therapy in the postoperative setting for intermediate andDocket No. 31134 / 2023-456 / PC high-risk thyroid cancer. With the renaissance of radiotheranostics, TSHR has been reconceived as an imaging and therapy target for thyroid diseases, including cancer; a few studies have explored and demonstrated TSHR as a target for nuclear imaging of thyroid cancer in preclinical animal models. These studies focused on radiolabeled TSHR agonists, including recombinant human TSH and its analogues TR1401 and TR1402, with radioactive iodine, Tc-99m, and Zr-89 for SPECT or PET imaging of thyroid cancer

[0012] , [15, 16], These imaging results are encouraging, but may be improved given the reported high TSHR expression on thyroid cancer. TSHR can exist in different states of affinity to its endogenous ligand TSH, and an agonist ligand is more sensitive to the high abundance of TSH, which binds TSHR

[0029] , Unlike agonists, which bind TSHR preferentially to the high-affinity state, antagonists bind TSHR in both high- and low-affinity states. Cryo-electron microscopy (cryo-EM) analysis showed that the TSHR antagonist human monoclonal antibody K1-70 bound TSHR extracellular domain with high binding affinity. Given the clear structure and mechanism of K1-70 binding TSHR complex, both specific TSHR radiotracers were designed by taking advantage of well-characterized K1-70 TSHR antagonist human monoclonal antibody as a lead ligand for Zr-89 and Cu-64 labeling, also disclosed in Parent, E. et al, Mol Imaging Biol. 2024 Oct; 26(5): 847-857, Fu, W. et al., Mol Pharm. 2025 Jul 7;22(7):4056-4067, and Fu, W. et al., Bioconjug Chem. 2025 Jul 15, the contents of which are incorporated by reference in their entirety herein. Additionally, clinical studies have demonstrated a strong therapeutic potential of this K1-70 antibody in thyroid diseases, including thyroid cancer [18, 19], This antibody has been used to treat a patient with RAI- resistant advanced follicular thyroid cancer and Graves' disease with encouraging results

[0030] , and has been further radiolabeled with different therapeutic radionuclides including67Cu,177Lu-, and225Ac- for treating thyroid cancers. Clinical translation of these radiopharmaceuticals will allow for selecting and treating only patients expressing tumor TSHR for precision therapy using radioligands.

[0046] Provided herein are thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceuticals, comprising a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide.TSHR-targeting moieties

[0047] To diagnose or treat diseases and disorders, the thyroid stimulating hormone receptor (TSHR)- targeting radiopharmaceuticals of the disclosure comprise a TSHR-targeting moiety. The ideal TSHR-targeting moiety should target and bind to the thyroid-stimulating hormone receptor (also called the thyrotropin receptor) expressed in a target tissue (e.g. cancerous tissue). In various embodiments, the TSHR-targeting moiety is a small molecule, a protein, a peptide, an antibody, a single-chain variable fragment (scFv), or an antibody fragment (Fab), any of which are capable of binding to the thyroid stimulating hormone receptor. In some cases, the TSHR-targeting moiety is a small molecule. In some cases, the TSHR-targeting moiety is a protein. In some cases, the protein is a non-antibody protein. In some cases, the TSHR-targeting moiety is a peptide. In some cases, the TSHR-targeting moiety is an antibody. In some cases, the TSHR-targeting moiety is a single-chainDocket No. 31134 / 2023-456 / PC variable fragment (scFv). In some cases, the TSHR-targeting moiety is an antibody fragment (Fab). In some cases, the TSHR-targeting moiety is an antibody or an scFv or a Fab thereof. In some cases, the TSHR- targeting moiety is an antibody or an scFv thereof.

[0048] Non-limiting examples of TSHR-targeting peptides include TSHR epitope small peptides or their analogs, small peptides P10 (FNPCEDIMGY, SEQ. ID NO. 2) and 9B-N (GLKMFPDLTKVYSTD, SEQ. ID NO. 3), and cyclic small peptides (amino acid sequence: CHQEEDFRVTC, SEQ. ID NO. 1).

[0049] Non-limiting examples of TSHR-targeting small molecules include any small molecule capable of binding to TSHR with nanomolar affinity, such as Org 274179-0 (CAS No. 1421683-12-6), or any of:Docket No. 31134 / 2023-456 / PC

[0050] In various embodiments, the TSHR-targeting moiety comprises an antibody (e.g., a mAb) specific for human thyroid-stimulating hormone receptor (i ,e. , a TSHR-targeting antibody). Suitable TSHR-targeting antibodies will be known to those in the art. In some cases, the antibody is a naturally-occurring antibody. In some cases, the antibody is an antagonist or an agonist. In some cases, the antibody is an antagonist. In some cases, the antibody is an agonist. In some cases, the antibody is a monoclonal antibody, a humanized antibody, or a human antibody. In some cases, the antibody is a monoclonal antibody. In some cases, the antibody is a humanized antibody. In some cases, the antibody is a human antibody. Non-limiting examples of suitable antibodies include K1 -70, Human Anti-TSHR Recombinant Antibody (clone K1 -70), K1-18, M22, or an scFv or a Fab thereof. In some cases, the antibody is K1-70, Human Anti-TSHR Recombinant Antibody (clone K1-70), K1- 18, M22, or an scFv thereof. In some cases, the antibody is K1-70, K1-18, or M22. In some cases, the antibody is K1-70. In some cases, the antibody is Human Anti-TSHR Recombinant Antibody (clone K1-70). In some cases, the antibody is K1-18. In some cases, the antibody is M22. In some cases, the antibody is an scFv of any of the antibodies disclosed herein. In some cases, the antibody is a Fab of any of the antibodies disclosed herein.

[0051] While the antibodies disclosed herein show TSHR-targeting utility as "naked” antibodies, the present disclosure focuses on their ability to form diagnostic and therapeutic agents, preferably diagnostic and therapeutic radiopharmaceuticals (radioconjugates), that target e.g., cancers expressing a cancer antigen.

[0052] 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., having a half-life of a day or more) preferable for chelation.Chimeric / Humanized Antibodies

[0053] Monoclonal antibodies may be modified for use as therapeutics or diagnostics. One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and / or light (L) chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is / are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. AlsoDocket No. 31134 / 2023-456 / PC included are fragments of such antibodies, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81 :6851-55.

[0054] 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.

[0055] 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).

[0056] The disclosure also provides for "derivatives” of antibodies contemplated herein, which term includes those proteins encoded by truncated or modified genes to yield molecular species functionally resembling the Ig fragments.

[0057] 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 et 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, the hosts 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.

[0058] In another embodiment, a monoclonal antibody is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using methods described in the art (Jones et al., 1986, Nature 321 :522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36), by substituting at least a portion of a rodent complementarity-determining region for the corresponding regions of a human antibody.

[0059] Chimeric, CDR grafted, and humanized antibodies and / or antibody variants are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein. In one embodiment, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described in the art.Docket No. 31134 / 2023-456 / PC

[0060] Also encompassed by the disclosure are human antibodies and antibody variants (including antibody fragments) that bind TSLP. Using transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production such antibodies are produced by immunization with a polypeptide antigen (i.e. , having at least 6 contiguous amino acids), optionally conjugated to a carrier. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. See also PCT App. Nos. PCT / US96 / 05928 and PCT / US93 / 06926. Additional methods are described in U.S. Pat. No. 5,545,807, PCT App. Nos. PCT / US91 / 245 and PCT / GB89 / 01207, and in European Patent Nos. 546073B1 and 546073A1. Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described in the art.Chelator Moiety

[0061] The TSHR-targeting radiopharmaceuticals of the disclosure comprise a chelator moiety capable of binding to a radionuclide. In particular, the chelator moiety is covalently attached to the TSHR-targeting radiopharmaceuticals of the disclosure, and allow the radiopharmaceuticals to be labeled with the radionuclide.

[0062] A variety of suitable chelating molecules for use in the chelator moieties disclosed herein are known to those skilled in the art. Non-limiting examples of such chelators include those based on desferrioxamine (DFO), 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA), sarcophagine (Sar), and derivatives thereof such as 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic amide (DOTAM), MECOSAR, BisCOSAR, AmBaSar (described e.g., in Cai, H., et al., Dalton Transactions 2009, (27), 5395-5400, incorporated herein by reference in its entirety) and DiBaSAr (described e.g., in US 2010 / 0196271, incorporated herein by reference in its entirety). In some cases, the chelator moiety comprises desferrioxamine (DFO), 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA), 1,4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic amide (DOTAM), or sarcophagine (Sar), or a derivative thereof. In some cases, the chelator moiety comprises desferrioxamine (DFO), 1,4,7- Triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA), or sarcophagine (Sar). In some cases, the chelator moiety comprises desferrioxamine (DFO). In some cases, the chelator moiety comprises 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA). In some cases, the chelator moiety comprises 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA). In some cases, the chelator moiety comprises 1,4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic amide (DOTAM). In some cases, the chelator moiety comprises sarcophagine (Sar). In some cases, the chelator moiety comprises a bifunctional chelator.

[0063] A chelator moiety disclosed herein can be directly linked to the TSHR-targeting moiety of the TSHR- targeting radiopharmaceutical, or it can be indirectly linked to the TSHR-targeting moiety. In some cases, the chelator moiety comprises a linker moiety, a prosthetic moiety, or both. In some cases, the chelator moiety comprises a chelating molecule indirectly linked to the TSHR-targeting moiety of the TSHR-targetingDocket No. 31134 / 2023-456 / PC radiopharmaceutical with a linker moiety. In some cases, the linker moiety comprises an alkyl chain, a polyalkylene oxide chain (e.g., a PEG chain), a peptide, or the like.

[0064] The chemistry, stoichiometry, and chemical structure used to attach the chelator moiety to the TSHR- targeting moiety (e.g. an antibody) can also be selected by those skilled in the art to achieve the desired radiopharmaceutical. For example, the chelator moiety can comprise a suitably reactive functional group that allows the chelator moiety to covalently bond with a portion of the TSHR-targeting moiety (e.g., an amino acid side chain of an antibody). Various functional groups have been used to conjugate a chelator to a biological moiety for radiolabeling, including isothiocyanate and N-hydroxysuccinimide (NHS) esters, isothiocyanate, or a maleimide moiety, which can be coupled to amines or sulfhydryl groups of proteins and peptides. For example, it is known that 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.

[0065] In some cases, the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, (DFO-Bn- SCN), p-SCN-Bn-NOTA, maleimido-mono-amide-NOTA, maleimido-mono-amide-DOTA, DOTA-NHS-ester, p- SCN-Bn-DOTA, p-SCN-Bn-DOTAM, DOTAM-NHS-ester, MECOSAR, BisCOSAR, AmBa-Sar, or DIBaSAr. In some cases, the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, (DFO-Bn-SCN), p-SCN- Bn-NOTA, maleimido-mono-amide-NOTA, maleimido-mono-amide-DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, AmBa-Sar, or DIBaSAr. In some cases, the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, p-SCN-Bn-NOTA, NOTA-NHS-ester, DOTA-NHS-ester, p-SCN-Bn-DOTAM, or p-SCN-Bn-DOTA. In some cases, the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, p-SCN-Bn-NOTA, NOTA-NHS- ester, DOTA-NHS-ester, or p-SCN-Bn-DOTA. In some cases, the chelator moiety comprises desferrioxamine-p- benzyl-isothiocyanate, (DFO-Bn-SCN). In some cases, the chelator moiety comprises p-SCN-Bn-NOTA. In some cases, the chelator moiety comprises maleimido-mono-amide-NOTA. In some cases, the chelator moiety comprises maleimido-mono-amide-DOTA. In some cases, the chelator moiety comprises DOTA-NHS-ester. In some cases, the chelator moiety comprises p-SCN-Bn-DOTA. In some cases, the chelator moiety comprises p- SCN-Bn-DOTAM. In some cases, the chelator moiety comprises DOTAM-NHS-ester. In some cases, the chelator moiety comprises MECOSAR. In some cases, the chelator moiety comprises BisCOSAR, In some cases, the chelator moiety comprises AmBa-Sar. In some cases, the chelator moiety comprises DIBaSAr. In some cases, the chelator moiety comprises AmBa-Sar-NHS ester. In some cases, the chelator moiety comprises DIBaSAr-NHS ester or NHS-DIBaSar-NHS ester. In some cases, the chelator moiety comprises MECOSAR-NHS ester. In some cases, the chelator moiety comprises BisCOSAR-NHS ester or NHS- BisCOSAR-NHS ester.

[0066] In some embodiments, the chelator comprises a bifunctional reagent for labeling TSHR-binding molecules. In some embodiments, the chelator moiety comprises N-Succinimidyl 4-[18F]fluorobenzoate (18F- SFB), N-succinimidyl 3-[131l]iodobenzoate (131I-SIB), or N-succinimidyl 3-[211At]astatobenzoate (211At-SAB). In some embodiments, the chelator comprises N-succinimidyl 4-[18F]fluorobenzoate (18F-SFB) or N-succinimidyl 3- [211At]astatobenzoate (211At-SAB). In some embodiments, the chelator comprises N-succinimidyl 4-Docket No. 31134 / 2023-456 / PC[18F]fluorobenzoate (18F-SFB). In some embodiments, the chelator comprises N-succinimidyl 3- [131l]iodobenzoate (131l-SI B). In some embodiments, the chelator comprises N-succinimidyl 3- [211At]astatobenzoate (211At-SAB).Radionuclides

[0067] The TSHR-targeting radiopharmaceuticals of the disclosure comprise one or more radionuclides. The term "radionuclide” as used herein refers to radioisotopes of atoms 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 several 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, beta particle, or Auger electron) from the nucleus. During those processes, the radionuclide 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.

[0068] In some cases, the radionuclide is a positron-emitter, an a-emitter, a p-emitter, an auger emitter, or a y- emitter. In some cases, the radionuclide is an a-emitter, a p-emitter, or an auger emitter. In some cases, the radionuclide is an a-emitter or a p-emitter. In some cases, the radionuclide is a positron-emitter or a y-emitter. In some cases, the radionuclide is a positron-emitter. In some cases, the radionuclide is an a-emitter. In some cases, the radionuclide is a p-emitter. In some cases, the radionuclide is an auger emitter. In some cases, the radionuclide is a y-emitter.

[0069] Nonlimiting examples of various radionuclides useful herein include, but are not limited to,111ln,125l,123l,201Ti,11C, AI-18F,89Zr,124l,68Ga,64Cu,61Cu,52Mn,55Co,44Sc,86Y,18F,149Tb,225Ac,211At,149Tb,212Pb,131l,177Lu,90Y,47Sc,188Re,67Cu,123l, or99mTc. In some cases, the radionuclide is89Zr,124l,68Ga,64Cu,61Cu,52Mn,55Co,44Sc,86Y,11C, or18F. In some cases, the radionuclide is89Zr or64Cu. In some cases, the radionuclide is225Ac,211At,149Tb, or212Pb. In some cases, the radionuclide is131l,177Lu,161Tb,90Y,47Sc,188Re, or67Cu. In some cases, the radionuclide is131l,177Lu,90Y,47Sc,188Re, or67Cu. In some cases, the radionuclide is123l or99mTc. In some cases, the radionuclide is111In. In some cases, the radionuclide is125l. In some cases, the radionuclide is123l. In some cases, the radionuclide is201Ti. In some cases, the radionuclide is123l. In some cases, the radionuclide is11C. In some cases, the radionuclide is AI-18F. In some cases, the radionuclide is89Zr. In some cases, the radionuclide is124l. In some cases, the radionuclide is68Ga. In some cases, the radionuclide is64Cu. In some cases, the radionuclide is61Cu. In some cases, the radionuclide is52Mn. In some cases, the radionuclide is55Co. In some cases, the radionuclide is44Sc. In some cases, the radionuclide is86Y. In some cases, the radionuclide is18F. In some cases, the radionuclide is225Ac. In some cases, the radionuclide is211At. In some cases, the radionuclide is149Tb. In some cases, the radionuclide is212Pb. In some cases, the radionuclide is131l. In some cases, the radionuclide is161Tb. In some cases, the radionuclide is177Lu. In some cases, the radionuclide is90Y. In some cases, the radionuclide is47Sc. In some cases, the radionuclide is188Re.Docket No. 31134 / 2023-456 / PCIn some cases, the radionuclide is67Cu. In some cases, the radionuclide is123l. In some cases, the radionuclide is99mTc.

[0070] Nonlimiting examples of the TSHR-targeting radiopharmaceuticals disclosed herein include89Zr-DFO- TSHR-Ab,64Cu-NOTA-TSHR-Ab,64Cu-NOTA-TSHR-Fab, and64Cu-NOTA-TSHR-scFv for immuno-PET imaging of TSHR expression. Biological evaluation in both cells and animal models showed significantly higher radiotracer uptake in TSHR-expressing tumors compared to TSHR-negative tumors in this proof-of-principle imaging agent development study. In addition, the coinjection of89Zr-DFO-TSHR-Ab,64Cu-NOTA-TSHR-Ab,64Cu-NOTA-TSHR-Fab, or64Cu-NOTA-TSHR-scFv with TSHR antibody enhances PET imaging of TSHR expression with increased tumor uptake and retention, as well as decreased background uptake. The data presented herein demonstrate that89Zr-DFO-TSHR-Ab,64Cu-NOTA-TSHR-Ab,64Cu-NOTA-TSHR-Fab, and64Cu-NOTA-TSHR-scFv are promising radiopharmaceuticals for PET imaging of TSHR-positive thyroid cancers. Additionally, the high tumor uptake and retention of therapeutic radiopharmaceuticals including67Cu-NOTA- TSHR-Ab,177Lu-DOTA-TSHR-Ab, and255Ac-DOTA-TSHR-Ab, as well as a low background in other major organs, demonstrate that the TSHR has potential as a biological target for radiotheranostic development for combined thyroid cancer imaging and therapy.

[0071] Additional TSHR-targeting radiopharmaceuticals of the disclosure include those listed in Table 1.Table 1Docket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCDocket No. 31134 / 2023-456 / PCPharmaceutical and Therapeutic Compositions and Their Administration

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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 suchDocket No. 31134 / 2023-456 / PC pharmaceutical compositions may be found, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton Pennsylvania (Gennaro 18th ed. 1990).

[0076] 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.

[0077] 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.

[0078] 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 the circulatory 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.

[0079] Other pharmaceutically acceptable carriers for polypeptide compositions of the present disclosure are liposomes, pharmaceutical compositions in which the active agent is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active agent 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.Docket No. 31134 / 2023-456 / PC

[0080] 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.

[0081] 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

[0082] The radioisotope constructs described in this disclosure are useful for diagnostics or therapeutics against a disease or disorder. The constructs for diagnosis and / or therapy could 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 different entities (e.g. Ga-68 for imaging and Lu-177 for therapy) depending on the type of radioisotopes used.

[0083] Further provided herein are methods of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a therapeutically-effective amount of a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide. In some cases, the TSHR-targeting radiopharmaceutical is a TSHR-targeting radiopharmaceutical disclosed herein, e.g., a TSHR-targeting radiopharmaceutical listed in Table 1.

[0084] In some cases, the disease or disorder is cancer or a thyroid disease. In some cases, the disease or disorder is cancer. In some cases, the cancer is thyroid cancer. In some cases, the cancer is differentiated thyroid cancer, papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), insular thyroid carcinoma (ITC), anaplastic thyroid carcinoma (ATC), medullary thyroid carcinoma (MTC), or oncocytic thyroid carcinoma (OTC). In some cases, the cancer is one or more of differentiated thyroid cancer (DTC), papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), insular thyroid carcinoma (ITC), anaplastic thyroid carcinoma (ATC), or oncocytic thyroid carcinoma (OTC). In some cases, the cancer is differentiated thyroid cancer (DTC). In some cases, the cancer is papillary thyroid carcinoma (PTC). In some cases, the cancer is poorly differentiated thyroid carcinoma (PDTC). In some cases, the cancer is insular thyroid carcinoma (ITC). In some cases, the cancer is anaplastic thyroid carcinoma (ATC). In some cases, the cancer is medullary thyroid carcinoma (MTC). In some cases, the cancer is oncocytic thyroid carcinoma (OTC). InDocket No. 31134 / 2023-456 / PC some cases, the cancer is metastatic cancer. In some cases, the disease or disorder is a thyroid disease. In some cases, the thyroid disease is Graves disease or hyperthyroidism. In some cases, the thyroid disease is Graves disease. In some cases, the thyroid disease is hyperthyroidism.

[0085] Also provided herein are methods of treating a disease or disorder in a patient, comprising the steps of: (a) diagnosing a disease or disorder in a patient, comprising i) administering to the patient a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide;(ii) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET);(ill) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater; and b) treating the disease or disorder by administering to the patient a therapeutically-effective amount of the thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical.

[0086] In some cases, the measuring comprises Single Photon Emission Computed Tomography (SPECT). In some cases, the measuring comprises Positron Emission Tomography (PET). In some cases, the first tissue is tumor tissue. In some cases, the second tissue is liver tissue, muscle tissue, or blood. In some cases, the second tissue is liver tissue. In some cases, the second tissue is muscle tissue. In some cases, the second tissue is blood.

[0087] A method of treating a disease includes administering a therapeutically effective amount of a radiopharmaceutical, e.g., thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceuticals of the disclosure. The administration may be given once or may be given in one to six cycles with a cycle duration from 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.

[0088] 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 therapeutic agent.

[0089] 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 of64Cu-NOTA-TSHR-Ab may beDocket No. 31134 / 2023-456 / PC administered from 1 week to 12 weeks and preferably 4 to 8 weeks following administration of a therapeutic dose of67Cu-NOTA-TSHR-Ab to assess the efficacy of the therapeutic dose on the disease. In some embodiments, such as64Cu-NOTA-TSHR-Ab for example, a subsequent therapeutic dose can be imaged to assess efficacy of a prior one.Cancer

[0090] The TSHR-targeting radiopharmaceuticals of the present disclosure are useful in the treatment of cancer, especially thyroid cancer.

[0091] Thyroid cancer is projected to become the fourth leading type of cancer across the world, and the causes appear to be complex and multifactorial, including dietary changes and environmental exposures.[1] Patients with thyroid cancer have widely different clinical outcomes depending on the pathological subtype and mutation profile. Advanced thyroid cancers, such as poorly differentiated thyroid cancer, anaplastic thyroid cancer, as well as metastatic follicular, papillary, and Hurthle cell thyroid cancers (the new terminology is oncocytic thyroid carcinoma, OCT), are aggressive and hard to treat, and often refractory to radiation ablation including radioiodine therapy and other standard therapies. [3| OTC, also known as Hurthle cell carcinoma, is the fourth most common of all thyroid cancers and was reclassified as a distinct entity in 2022 by the World Health Organization.

[0037] OTC typically has a more aggressive clinical behavior compared to other differentiated thyroid cancers with a higher rate of distant metastases.

[0038] The diagnosis of OTC has typically relied on histopathology, and the presence of capsular and / or vascular invasion are hallmarks of OTC, and typically diagnosed after thyroid lobectomy or total thyroidectomy.

[0039] Total thyroidectomy is typically performed in cases that would suggest utilization of iodine-131 Nal (131l) therapy and thyroidectomy one of the few independent predictors of improved cancer-specific survival.

[0040]

[0092] Postoperative radioactive iodine (RAI) has dual roles for the diagnosis and treatment of metastatic thyroid cancer. The lower energy SPECT isotope 1-123 Nal (123l) is routinely used for imaging and treatment planning, and the strong |3 emitter,1311, is used to ablate residual physiologic thyroid tissue and to also treat metastatic sites of OTC. [41 , 42] Unfortunately, advanced thyroid cancer, such as OTC, has high false-negative radioiodine uptake. Compared to other differentiated thyroid carcinomas, with reported false negative rates between 33% and 99% depending on the site of disease

[0043] , and only about 10% patients with OTC metastases take up1311, resulting in the limited efficacy of1311 therapy.

[0044] For example, a study of 239 patients found no difference in cancer-specific survival in patients with OTC that received RAI compared to those who did not.

[0042] However, a survey of the National Cancer Data Base found that patients with high risk OTC did find see an improvement in survival in patients who received131l therapy compared to those without131l.

[0041] 2-[18F]fluoro-2- deoxy-D-glucose (FDG) PET / CT is a clinically utilized alternative to imaging of patients with low radioiodine avidity and in presumed or known metastatic thyroid cancer. [45, 46] FDG PET has been reported to have both a high sensitivity and specificity, but there are known false-positives (e.g inflammation, infection) and false negative (e.g. radioiodine avid disease) both findings commonly seen in thyroid cancer.

[0047] While there are a few reportsDocket No. 31134 / 2023-456 / PC of other molecular imaging tracers, such as18F-fluorodihydroxyphenylalanine ([18F]FDOPA)

[0048] and68Ga- DOTATATE

[0049] that have incidentally identified OTC, and clinical trials investigating radiotheranostic agents targeting somatostatin receptor type 2 (SSTR2) in OTC, including68Ga-DOTATATE (NCT04927416) and177Lu- DOTA-EB-TATE (NCT06991738), are currently ongoing. However, metastatic thyroid cancer remains a challenging disease to treat. Therefore, novel diagnostic and therapeutic agents for patients with metastatic thyroid cancer are highly needed.

[0093] Thyroid-stimulating hormone receptor (TSHR) activation is a major regulator of thyroid function and growth, and both mRNA and protein expression of TSHR are largely limited to the thyroid gland and thyroid tumors. [6] Thyroid-stimulating hormone (TSH), via its receptor TSHR, promotes expression of downstream effector genes to augment growth, differentiation and thyroid hormone synthesis, and secretion of thyroxine (T4) and triiodothyronine (T3)

[0050] , Activation of downstream signaling cascades is the primary route of de novo carcinogenesis and growth of thyroid carcinomas. [38, 51] TSHR is predominately expressed on the basolateral membrane of thyroid follicular cells

[0052] and is highly expressed in well differentiated thyroid cancer, such as OTC, but is less well expressed in poorly differentiated cell types. [6] An immunohistochemical study showed TSHR overexpression in approximately 62% of OTC.

[0053] While TSH suppression has been shown to attenuate OTC growth, precision TSH suppression therapy is often suboptimal as only a subset of patients will respond to therapy and there are well-known comorbid conditions. [54, 55]

[0094] Chimeric antigen receptor (CAR)-T cell therapy has been shown to have remarkable clinical response in various malignancies such as lymphoma and multiple myeloma

[0056] , CARs are designed receptors that redirect lymphocytes, primarily T-cells, to target and kill cells expressing a specific target antigen and can result in exceptionally strong anti-tumor responses. While the promises of CAR-T therapy may revolutionize individualized medicine for cancer patients, there are several limitations to CAR-T therapy that need to be addressed including: reduced efficacy against solid tumors, poor tumor infiltration, and limited prognostic tools to identify which patients will respond best to CAR-T therapy. To this end, TSHR CAR-T cell therapy has been developed to treat patients with RAI-refractory disease who would otherwise die due to failure of all standard-of- care therapies. [57, 58] TSHR-expressing advanced thyroid tumors are known to be commonly infiltrated by immune cells, including T cells. [2] A recent study reported a complete response (CR) and a near CR (74%) in two patients with high metastatic burden thyroid cancer to multiple organs and RAI resistance.

[0059] This limited data suggests potential feasibility and efficacy of TSHR CAR-T cell therapy in patients with deadly advanced metastatic thyroid cancer, including metastatic OTC.

[0058] However, as noted earlier, there is a subset of patients with advanced thyroid cancer which demonstrate attenuated or no TSHR protein expression, and thus would not be candidates for TSHR targeted CAR-T therapy, necessitating prognostic whole body interrogation of TSHR expression prior to TSHR directed CAR-T therapy [60, 61],

[0095] Given its overexpression in well differentiated thyroid carcinomas such as OTC, targeted imaging of TSHR has the potential to improve upon metastatic thyroid cancer detection and help guide clinical management, including TSHR targeted CAR-T therapy. Recently, an in-vitro evaluation of a novel TSHR agonist was reportedDocket No. 31134 / 2023-456 / PC to have accumulation in TSHR positive thyroid cancer cells and demonstrated adequate biodistribution and uptake in TSHR expression tumors.

[0017] A novel Zr-89 labeled TSHR specific human monoclonal autoantibody has also been synthesized, K1-70 (89Zr-DFO-TSHR-Ab), for immuno-PET imaging of TSHR expression.

[0062] Published data have demonstrated that89Zr-DFO-TSHR-Ab is a promising radiopharmaceutical for PET imaging of TSHR-positive thyroid cancers. Due to the highly aggressive nature of advanced thyroid cancer and low efficacy of131l therapy, the utility of89Zr-DFO-TSHR-Ab PET imaging has been explored in preclinical tumor cell line XTC-UC1 ,

[0063] derived from a metastatic OTC, retains TSHR expression as well as other markers of differentiation and was evaluated as a target of89Zr-DFO-TSHR-Ab uptake in both in vitro and mouse animal models. Additionally,89Zr-DFO-TSHR-Ab PET has been evaluated as a prognostic agent and therapeutic monitor of TSHR CAR-T cell therapy in the OTC tumor mouse model.Cold Kits

[0096] Targeted antigens, such as TSHR, often have low-level expression in 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.

[0097] 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).

[0098] Importantly, because the antibody mass dose for a radiolabeled antibody for imaging or therapeutic applications can typically be quite small (e.g. < 5mg or < 5pig radiolabeled antibody), antibody radiopharmaceuticals are susceptible to TMDD.

[0099] 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.

[0100] 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.

[0101] 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 theDocket No. 31134 / 2023-456 / PC20 - 50mg range. (Lohrmann et al., Clin Cancer Res 2019;25:7014-23 and Mortimer et al., J Nucl Med 2014; 55:23-29)

[0102] 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.

[0103] It therefore 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.

[0104] For example, the final drug product may include the radiolabeled antibody, such as89Zr-DFO-TSHR- Ab,64Cu-NOTA-TSHR-Ab, or other radioisotopes and linkers described herein, 0.05 mg - 100 mg of cold TSHR- Ab, 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, and others known to those skilled in the art.

[0105] In various embodiments, the amount of unconjugated "cold” antibody, such as TSHR-Ab, administered may be from 0.05 mg to 100 mg and preferably from 1.0 mg to 40 mg. The timing of cold (unlabeled) antibody administration may be pre-dosing, post-dosing, or concurrent dosing of the radiolabeled "hot” antibody, such as89Zr-DFO-TSHR-Ab or64Cu-NOTA-TSHR-Ab. In some cases, the cold antibody is an unlabeled TSHR-targeting antibody. In some cases, the unlabeled TSHR-targeting antibody is K1-70.

[0106] The cold antibody may also be included in a kit comprising the final drug product vial or other acceptable container of the formulation89Zr-DFO-TSHR-Ab,64Cu-NOTA-TSHR-Ab, or other TSHR-Ab compounds or constructs described herein in an appropriately shielded container, a vial or other acceptable container of cold TSHR-Ab 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.

[0107] 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.Docket No. 31134 / 2023-456 / PC

[0108] 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.

[0109] Using the antibody-based radioisotope constructs described in this disclosure, the construct could be administered, and one or more pharmacokinetic measurements could 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.

[0110] Pharmacokinetic (PK) parameters such as the Area under the curve (AUG), 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.

[0111] 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 could be compared to a preferred curve and then the antibody mass dose could be increased or decreased.

[0112] Additional or fewer timepoints could also be assessed. For example, the amount of radiopharmaceutical in the blood at a single timepoint such as an imaging scan between 0.5 and 10, preferably between 1 and 3 days after injection of an imaging agent (non-limiting examples of which include89Zr-DFO- TSHR-Ab) could be assessed to determine if a sufficient quantity of drug, such as at least approximately 10% injected dose per gram (10% ID / g), remains in circulation. If the circulating dose is significantly higher than the target (e.g. 30% ID / g in the blood), the total antibody mass dose for a subsequent administration such as a therapeutic administration (non-limiting examples include64Cu-NOTA-TSHR-Ab) would be decreased, and if the circulating dose is lower than the target 10% ID / g, the antibody mass dose would be increased. For administration of TSHR-Ab, a total antibody mass dose ranging from 0.5 mg - 100 mg could be used, which covers the dosing range where TMDD typically has a strong effect.

[0113] 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.

[0114] 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. Radiosentsitization can be achievedDocket No. 31134 / 2023-456 / PC by 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.

[0115] Other chemotherapeutic agents, CAR T-cell therapies, etc. could also be used to target multiple cancer-causing and progressing pathways. These drugs could be co-administered or administered one after the other or compatible drugs could be assembled in a single or multiple delivery system.

[0116] The radiopharmaceutical could also be paired with external radiotherapy to improve efficacy.

[0117] To improve the distribution of the antibody radioisotope constructs, use of blood flow modulators such as vasodilators like acetazolamide could also be employed.Methods of Diagnosis

[0118] Also provided herein are methods of diagnosing a disease or disorder in a patient, comprising the steps of:(a) administering to the patient a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide;(b) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours 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 2:1 or greater. In some cases, the TSHR-targeting radiopharmaceutical is a TSHR-targeting radiopharmaceutical disclosed herein, e.g., a TSHR-targeting radiopharmaceutical listed in Table 1. In some cases, the first tissue is tumor tissue. In some cases, the second tissue is liver tissue, muscle tissue, or blood.In some cases, the second tissue is liver tissue. In some cases, the second tissue is muscle tissue. In some cases, the second tissue is blood.

[0119] In some cases, the disease or disorder is cancer or a thyroid disease. In some cases, the disease or disorder is cancer. In some cases, the cancer is thyroid cancer. In some cases, the cancer is one or more of differentiated thyroid cancer, papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC),Docket No. 31134 / 2023-456 / PC insular thyroid carcinoma (ITC), anaplastic thyroid carcinoma (ATC), or oncocytic thyroid carcinoma (OTC). In some cases, the cancer is differentiated thyroid cancer. In some cases, the cancer is papillary thyroid carcinoma (PTC). In some cases, the cancer is poorly differentiated thyroid carcinoma (PDTC). In some cases, the cancer is insular thyroid carcinoma (ITC). In some cases, the cancer anaplastic thyroid carcinoma (ATC). In some cases, the cancer is oncocytic thyroid carcinoma (OTC). In some cases, the cancer is metastatic cancer. In some cases, the disease or disorder is a thyroid disease. In some cases, the thyroid disease is Graves disease or hyperthyroidism. In some cases, the thyroid disease is Graves disease. In some cases, the thyroid disease is hyperthyroidism.

[0120] A method of assessing the aggressiveness of disease includes administering a diagnostically effective amount of a TSHR-targeting radiopharmaceutical, 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 2:1 or greater. In some cases, the first tissue is tumor tissue. In some cases, the second tissue is liver tissue, muscle tissue, or blood. In some cases, the second tissue is liver tissue. In some cases, the second tissue is muscle tissue. In some cases, the second tissue is blood.Kits

[0121] 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 radiopharmaceutical 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 or multiple doses in the same vial. 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 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.

[0122] 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.

[0123] 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.Docket No. 31134 / 2023-456 / PCEXAMPLESMaterials and General Methods

[0124] Human Anti-TSHR Recombinant Antibody (clone K1-70), TSHR-Fab, and TSHR-scFv were purchased from Creative Biolabs, Inc. (NY, USA). The concentration of the antibody and its conjugate was measured by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The bifunctional chelators, desferrioxamine-p-benzyl- isothiocyanate (p-SCN-Bn-DFO) (catalog No. B-705), S-2-(4-lsothiocyanatobenzyl)-1 ,4,7,10- tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), and 2-S-(4-lsothiocyanatobenzyl)-1 ,4,7- triazacyclononane-1 ,4,7-triacetic acid (p-SCN-benzyl-NOTA), were purchased from Macrocyclics, Inc. (Dallas, TX, USA).89Zr-oxalate solution,64CuCl2 in 0.05 M aqueous solution were purchased from the Cyclotron Laboratory at the University of Wisconsin, Madison (Madison, Wl, USA). Water used for this study was ultrapure (> 18.2 MQcm-1 at 25 °C).67CuCl2 in 0.05 M aqueous solution was purchased from Idaho Accelerate center (Pocatello, ID, USA).225Ac and177Lu were purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA). Zeba Micro Spin Desalting Column 40k MWCO, 75 piL was ordered from Thermo Fisher Scientific. (Waltham, MA, USA). PD-10 gel filtration Columns (GE Healthcare) and other chemicals were purchased from Millipore Sigma (Burlington, MA, USA) unless otherwise noted. AR-2000 radio-thin-layer chromatography (TLC) scanner (Eckert& Ziegler, Wilmington, MA, USA) and ITLC-SG paper (Agiient Technologies, Santa Clara, CA) were used to perform TLC analysis.TSHR antibody K1-70 conjugation and radiolabeling

[0125] The conjugation of chelator p-SCN-Bn-DFO, p-SCN-Bn-DOTA, or p-SCN-Bn-NOTA to TSHR antibody K1-70 or its Fab or scFv, followed by radiolabeling with Zr-89, Ou-64, Ou-67, Lu-177, or Ac-225 were prepared with minor modifications as described in the literature [17, 24],Antibody conjugation

[0126] The conjugation of chelator p-SCN-Bn-DFO, p-SCN-Bn-DOTA, or p-SCN-benzyl-NOTA to TSHR antibody K1-70, its Fab, or scFv was carried out as described below. In brief, 1 mg (7 nmol) of antibody K1-70 in 1 mL PBS solution (pH 7.4) was concentrated to 0.3 mL using an Amicon Ultra-15 centrifuge filter (10K Da cut off) and transferred to a 1.5 mL Eppendorf tube, followed by adding 0.1 M Na2CO3 buffer to adjust TSHR K1-70 antibody solution to pH 8.9-9.1. The 2.1 piL solution of p-SCN-Bn-DFO (21 nmol) dissolved in DMSO (7.53 mg / mL) was added to the TSHR K1-70 solution. This formed a three-fold molar excess of the chelator over the molar amount of K1-70 and was mixed immediately for conjugation. The mixture was incubated at 37 °C for 120 minutes using a Thermomixer at 500 r.p.m., followed by purification with a Zeba spin desalting column (0.5 mL) with a 40K or 7K molecular weight cut-off filtration using 1 M HEPES buffer (pH 7.4) or 0.2 M NH4AC buffer (pH 6.5) as the eluent to remove excess chelator. The purified TSHR antibody conjugate, DFO-TSHR-Ab, DOTA- TSHR-Ab, NOTA-TSHR-Fab, NOTA-TSHR-scFv, or NOTA-TSHR-Ab was collected. The UV-Vis spectrophotometer determined the concentration of DFO-TSHR-Ab, DOTA-TSHR-Ab, NOTA-TSHR-Fab, NOTA- TSHR-scFv, or NOTA-TSHR-Ab as 3 mg / mL. The number of DFO, DOTA, or NOTA molecules attached to K1-Docket No. 31134 / 2023-456 / PC70, its Fab, or scFv was measured with a radiometric isotopic dilution assay. The conjugate DFO-TSHR-Ab, DOTA-TSHR-Ab, NOTA-TSHR-Fab, NOTA-TSHR-scFv, or NOTA-TSHR-Ab was stored in a -20 °C freezer and was found to be stable for more than 18 months for further radiolabeling use.Antibody conjugate radiolabeling

[0127] 89Zr-DFO-TSHR-Ab was prepared by complexing neutralized89Zr-oxalate with the conjugate DFO- TSHR-Ab as described below. Briefly,89Zr-oxalate (2.5 ± 0.1 mCi in 25-50 piL) was adjusted to pH 6.8-7.5 using 1 .0 M HEPES and 1 .0 M Na2CO3 solution and incubated for 3 min at room temperature. The conjugate DFO- TSHR-Ab (80-100 pig) in 1 M HEPES buffer (pH 7.4) solution (30 piL) was added to the above neutralized89Zr- oxalate solution and incubated at 37 °C for 60 minutes for radiolabeling. The resulting89Zr-DFO-TSHR-Ab mixture was purified using a PD10 gel filtration column with 0.25 M sodium acetate (pH 5.4 - 5.6) with 5 mg / ml gentisic acid solution as eluent. The radiochemical identity and purity of the final product89Zr-DFO-TSHR-Ab were determined by radio-TLC with 50 mM EDTA (pH 8.0) as the developing solution.

[0128] The radiotracer, [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR- scFv, was prepared with slight modifications to a previously reported protocol.24Briefly, approximately 20-100 pig of purified NOTA-TSHR-Ab, NOTA-TSHR-Fab, or NOTA-TSHR-scFv in 0.2 M NH4Ac buffer (pH 6.5) was used for copper-64 labeling. A total of 370-740 MBq of [64Cu]CuCl2 (10-20 pL, 0.05 M HOI) was added to 200 pL of 0.1 M NH4Ac buffer (pH 5.5), followed by mixing with NOTA-TSHR-Ab, NOTA-TSHR-Fab, or NOTA-TSHR- scFv and incubation at 37 °C for 30-60 min in a Thermomixer set to 500 rpm. After completion of the radiolabeling reaction, the product was purified via a PD-10 desalting column using 0.9% sodium chloride or PBS elution, yielding [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR-scFv. The radiochemical identity and purity of the final product were determined by instant radio-TLC with 50 mM EDTA (pH 8.0) as the developing solvents and further characterized by radio-SEC-HPLC.

[0129] Therapeutic radiopharmaceuticals67Cu-NOTA-TSHR-Ab,177Lu-DOTA-TSHR-Ab, and225Ac-DOTA- TSHR-Ab were prepared with slight modifications to the above protocol. For67Cu labeling, ~60 pig of purified NOTA-TSHR-Ab in 0.2 M NH4Ac buffer (pH 6.5) was mixed with -740 MBq of67CuCl2, and the pH was adjusted to 5-6 using 0.1 M NH4Ac buffer (pH 5.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% sodium chloride, yielding67Cu-NOTA-TSHR-Ab. The radiochemical identity and purity of the final product were determined by instant radio-TLC with 50 mM EDTA (pH 8.0) as the developing solvents. For177Lu labeling, 60-90 pig of purified DOTA-TSHR-Ab in 0.01 M PBS (pH 7.4) was mixed with 30-100 MBq of177LuCl3 and ascorbic acid, and the pH was adjusted to 5-6 using 0.1 M NH4Ac buffer (pH 5.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% sodium chloride, yielding177Lu-DOTA-TSHR-Ab. The radiochemical identity and purity of the final product were determined by instant radio-TLC with 50 mM EDTA (pH 8.0) as the developing solvents. For225Ac labeling, -80 pg of purified DOTA-TSHR-Ab in 0.2 M NH4OAc (pH 6.5)Docket No. 31134 / 2023-456 / PC was mixed with ~3 MBq of225Ac3+and ascorbic acid, and the pH was adjusted to 5-6 using 0.2 M NH4OAC buffer (pH 6.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% sodium chloride, yielding225Ac-DOTA-TSHR-Ab. The radiochemical identity and purity of the final product were determined by instant radio-TLC with 50 mM EDTA (pH 8.0) as the developing solvents.Human cancer cell lines and in vitro cell uptake and binding studies

[0130] Two human thyroid cancer cell lines, THJ-529T and FTC-133, and human chronic myelogenous leukemia cell line K562 (null for TSHR) were used in the present studies. All three cell lines were transduced to stably express the TSHR gene, and their respective wild type (WT) parental cell line possesses low or no TSHR expression. The THJ-529T cell line was derived from a male patient with poorly differentiated thyroid carcinoma

[0025] . The FTC-133 cell line was derived from a male patient with differentiated follicular thyroid carcinoma (FTC)

[0027] ,Cell culture, in vitro cell uptake study, and cell binding assay

[0131] Human leukemia cell line K562 was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). All cell lines were cultured in RPM1 1640 medium (Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Cells stably expressing the TSHR were supplemented with 2-5pig / ml puromycin, antibiotic selection to retain TSHR expression (Selleckchem, Houston, TX, USA). TSHR expression was verified using flow cytometry performed on a three- laser CytoFLEX (Beckman Coulter, Chaska, MN, USA). Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. Short tandem repeat (STR; aka DNA fingerprinting) was performed on all cell lines by IDEXX BioAnalytics using CellCheckl 6 human Marker STR Profile and Inter-species Contamination Test. Identity matching score above 80% was consistent with cell line of origin.

[0132] A wild-type THJ529T (THJ529TM / T) cell line, derived from a male patient with poorly differentiated thyroid carcinoma, was studied. The TSHR-positive THJ529T (THJ529TreHfi+model was engineered to stably expresses the TSHR gene, whereas the parental cell line TH J529TM / rexhibits low or negligible TSHR expression.14’15Both the THJ529TreHR+and THJ529TM / rcells were cultured in RPM1 1640 medium containing 10% FBS and were grown in a humidified incubator at 37 °C with 5% CO2.

[0133] The imaging specificity and binding affinity of [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR-scFv were evaluated using TSHR expressing THJ529T cells or its wild type with low or no TSHR expression. THJ529Tl4 / 7' or THJ529TreHR+cells (2 x 105cells / well) were seeded into 24-well plates and incubated overnight. For the radiotracer uptake and the blocking assay, cells were divided into TH J529Tl4 / r, TH J529TreHfi+, and TH J529TreHR+co-incubated with non-radioactive TSHR-Ab (~500-fold excess of [64Cu]Cu- NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR-scFv, ~1-7 pg / well) groups. Each well was treated with 0.0185 MBq of [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA- TSHR-scFv and incubated at 37 °C for 60 min. For saturation assay, THJ529TreHR+cells were incubated withDocket No. 31134 / 2023-456 / PC increasing concentration of [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR- scFv (0-1.7 nM / well) at 37 °C for 60 min. In the competition assay, THJ529TreHR+cells were pre-saturated with varying concentrations of non-radioactive TSHR-Ab (0.04-10.00 pig / well) and subsequently incubated with 0.0185 MBq of [64Cu]Cu-NOTA-TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR-scFv per well at 37 °C for 60 min. Uptake in THJ529TreHR+cells over time and immunoreactivity assay of [64Cu]Cu-NOTA-TSHR- Ab, [64Cu]Cu-NOTA-TSHR-Fab, or [64Cu]Cu-NOTA-TSHR-scFv were also conducted.

[0134] All the cell experiments above were performed in triplicate. Following incubation, the cells were washed three times with DPBS, lysed with NaOH (1 M), and the lysates were collected. Radioactivity was measured using a y counter. The dissociation constant (Kd) and inhibition constant (Ki) were calculated using GraphPad Prism version 10.0.0 (CA, USA).Immunocytochemistry (ICC) for characterizing TSHR expression on tumor cells

[0135] Cells were washed three times with PBS (Corning, NY, USA), scraped from the bottom of the plate using the cell lifter (Fisherbrand, Waltham, MA, USA), transferred to 50 mL polypropylene the tube and centrifuged at room temperature for 2 min at 500 x g. The supernatant was aspirated, and 10 % neutral buffered formalin (Fisher Scientific, Waltham, MA, USA) was added for 30 min at room temperature. HistoGel (Thermo Scientific, Waltham, MA, USA) was heated in the microwave for 3 seconds at 100% power and converted to a liquid state. Cells were transferred to the HistoScreen Tissue Cassettes (Thermo Scientific, Waltham, MA, USA) and covered with liquid Histogel. Samples were placed at room temperature and allowed to solidify. HistoGel blocks were transferred into tissue embedding cassettes (Thomas Scientific, Swedesboro, NJ, USA) and dehydrated in increasing concentrations of ethanol, xylene, and paraffin embedding. Sections were prepared as described in the immunohistochemistry section. Slides were autoclaved for 15 min in a high pH 9.0 antigen retrieval buffer (Dako, Glostrup, Denmark). Primary antibody was used for 60 minutes at room temperature, antihuman: TSHR antibody at 1 :1500 (Abeam, ab218108, Cambridge, UK). Envision labeled polymer (Dako, Glostrup, Denmark) was used for 30 min as a secondary antibody. Slides were stained with diaminobenzidine tetrahydrochloride (DAB) chromogen (Dako, Glostrup, Denmark) for 5 min at room temperature and counterstained with Mayer's hematoxylin (Sigma-Aldrich, Burlington, MA, USA). Control staining with hematoxylin and eosin (Sigma-Aldrich, Burlington, MA, USA) was also performed. Aperio AT2 scanner (Leica, Wetzlar, Germany) was used for the digital image at 20x objective. All analyses were performed using FlowJo X10.0.7r2 software (Ashland, OR, USA). Images were analyzed using the Aperio ImageScope software (Aperio Technologies, Vista, CA, USA).Immunohistochemistry (IHC) characterizing TSHR expression on tumor tissues

[0136] Immunostaining was performed on paraffin-embedded tissue. Paraffin blocks were cut into 5 pm thick sections. Slides were deparaffinized, rehydrated with decreasing concentrations of ethanol, and finally placed in water. Antigen retrieval was performed using Antigen Retrieval Solution (pH 9) (Dako, Glostrup, Denmark) for at least 15 min in an autoclave, and cooled for 30 min at room temperature. The activity of endogenousDocket No. 31134 / 2023-456 / PC peroxidases was blocked by washing slides with 3% hydrogen peroxide (Fisher Scientific, Waltham, MA, USA) for 10 min. The primary antibody was used for 60 minutes at room temperature, anti-human: TSHR antibody at 1 :500 (Abeam, ab218108, Cambridge, UK). The primary antibody was visualized using the Envision Dual Labeled Polymer kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions. After washing, slides were counterstained with Gill I Hematoxylin (Sigma-Aldrich, Burlington, MA, United States). Slides were dehydrated through ethanol and xylene and cover-slipped using a xylene-based mounting medium (Fisher Scientific, Waltham, MA, USA). Samples were examined under bright-field illumination at x 20 objectives, and digital images were obtained using Aperio AT2 (Leica, Wetzlar, Germany). Results were processed using Aperio eSlide Manager and H-Score values were estimated using the Aperio ImageScope Software (Aperio Technologies, Vista, CA, USA). Normal tissues were used as positive and negative controls.

[0137] To validate the results of micro-PET / CT imaging of TSHR expression in tumor xenograft mouse models, ex vivo IHC for TSHR expression in tumors was performed. Tumor xenografts from THJ529TreHR+ / M / rmodels were collected, embedded in paraffin, and sectioned into 5-pim-thick slices. The sections were dewaxed using dimethylbenzene and rehydrated through a graded alcohol series. Antigen retrieval was achieved using a Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH 9.0). To minimize nonspecific binding, the slices were blocked with 5% BSA for 2 h at RT prior to incubation with primary antibody. A rabbit anti-human K1-70 primary antibody and a goat anti-rabbit IgG secondary antibody were used for staining.Tumor mouse models

[0138] All animal experimental procedures and studies were performed in accordance with approved protocol from the Institutional Animal Care and Use Committee (IACUC). To establish subcutaneous tumor models, 6 to 8 week old Nod SCID gamma (NSG) mice were obtained from Jackson Laboratory. Animals were housed in ventilated cages and provided food and water, ad libitum. TSHR-expressing tumors and their corresponding WT as control groups were induced on two flanks by subcutaneous injection of 5x106cells in a 200 piL cell suspension of a 1 :1 mixture of fresh media DBPS I GFR Matrigel, respectively. After injection, animals were monitored by general observation, and these tumors were measured twice weekly with calipers. The tumor volume was determined (width x height x length / 0.0523 = mm3) and with injections occurring ~ 100-200 mm3for PET imaging and biodistribution studies.Small animal PET / CT imaging

[0139] The tumor xenograft mice (n= 3) in each group were administered 100 piL of89Zr-DFO-TSHR-Ab (SOTS piCi, ~3-4.5 pig) solution with or without 100 pig of TSHR antibody K1-70 via lateral tail vein injection, followed by G8 micro-PET / CT (Sofie Biosciences, Culver City, CA) imaging at the desired time points (24, 48, 72 hours post-injection). Mice were anesthetized during the procedure with isoflurane: oxygen gas mixture. Each mouse PET / CT data were acquired via a 10-minutes static scan. These images were reconstructed and analyzed using the MIM software (Cleveland, OH). For each micro-PET / CT scan, regions of interest were drawn over the tumor, liver, kidney, muscle, and blood on decay-corrected whole-body coronal images to a standard uptake valueDocket No. 31134 / 2023-456 / PC(SUV). For the PET scan, the axial FOV was 9.5 cm, the transaxial FOV was 4.7 cm, with a detector element size of 1.8 mm x 1.8 mm x 7 mm. The peak absolute system sensitivity was >14%, the reconstructed resolution at center of FOV was1.4 mm, the average energy resolution was 18%, and the energy window range 150-650 keV. The total number of detector elements was 5408, the reconstruction algorithm used was 3D ML-EM, and the scan time was 10-20 min. For the CT scan, X-ray source was set to 50 kVp and 200 piA, the X-ray camera had 75 micron pixels, 14 bits, and a CMOS + Csl flat panel, with a camera speed of 24 fps. The field of view was 100 mm axial by 50 mm transaxial, with a scan time of less than 60 s.

[0140] Mice were administered with 2.22-5.55 MBq of [64Cu]Cu-NOTA-TSHR-Ab in 100-200 piL of 0.9% sodium chloride solution, followed by anesthesia with a 3% isoflurane / oxygen gas mixture, and then subjected to G8 micro-PET / CT imaging at pre-determined time points. Based on previous research,16100 pg of nonradioactive TSHR-Ab can effectively reduce background signal without affecting tumor uptake. Therefore, 100 pg of non-radioactive TSHR-Ab was included in the imaging procedure to achieve better imaging contrast. Briefly, to study the pharmacokinetics, six mice (3 males and 3 females) bearing both TH J529TM / r(left flank) and THJ529TreHR+(right flank) tumors underwent PET / CT imaging at 1, 2, 4, 18, 24, and 48 h p.i. (co-injected with 100 pig of non-radioactive TSHR-Ab). Based on this result, the minimum effective dose of TSHR-Ab was further optimized. Twelve mice (n = 3 per dose) were co-injected with the mixture of 100, 50, 25, or 0 pig of nonradioactive TSHR-Ab and the radiotracer, followed by PET / CT imaging at 18 h p.i. Furthermore, to verified TSHR imaging specificity, five mice with THJ529TreHR+tumors underwent [18F]FDG PET / CT imaging on the first day, followed by [64Cu]Cu-NOTA-TSHR-Ab PET / CT imaging on the second day. Data from PET / CT imaging were reconstructed and analyzed using MIM version 7.3.5 software (Cleveland, OH, USA). For each image, regions of interest (ROIs) were drawn over the tumor, liver, blood, bone, kidney, and muscle on PET images to calculate the maximum standardized uptake value (SUVmax).

[0141] Mice bearing THJ529TTSHR+tumors were administered with ~3.7 MBq of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-scFv in 100-200 pL of 0.9% sodium chloride solution, followed by anesthesia with a 3% isoflurane / oxygen gas mixture, and then subjected to G8 micro-PET / CT imaging at pre-determined time points (1, 4, 18, and 24 h p.i.). For blocking imaging, mice were injected unlabeled TSHR-Ab 1 day prior radiotracer injection. Data from PET / CT imaging were reconstructed and analyzed using MIM version 7.3.5 software (Cleveland, OH, USA). For each image, regions of interest (ROIs) were drawn over the tumor on PET images to calculate the maximum standardized uptake value (SUVmax).Biodistribution studies and Immunohistochemistry (I HC)

[0142] To validate the accuracy of micro-PET / CT imaging of TSHR expression in tumor xenografts mouse models, ex vivo radiotracer biodistribution experiments were also performed with direct tissue sampling after the PET / CT imaging at 72 hours post-injection. In brief, at the end of micro-PET / CT imaging, mice were sacrificed and dissected to evaluate the biodistribution of89Zr-DFO-TSHR-Ab. Samples of blood, muscle, liver, kidney, and tumors were removed and counted in the gamma counter. Tissues were weighed, and the radioactivity of theDocket No. 31134 / 2023-456 / PC tissue samples were calibrated against a known aliquot of the injected activity. The biodistribution results were presented as percentage of injected dose per gram of tissue (%ID / g) and then compared with micro-PET / CT imaging data. Immunostaining was performed on paraffin-embedded tumor samples to verify TSHR expression for each tumor and cell lines as previously described.

[0143] Six mice bearing both THJ529TreHR+and TH J529TM / rtumors were euthanized immediately after PET imaging of [64Cu]Cu-NOTA-TSHR-Ab for the 48-hour biodistribution study (n = 6), and another group of three mice xenografts were sacrificed for the 18-hour biodistribution study (n = 3). Additionally, twelve THJ529TreHR+tumor-bearing mice (n = 3 per dose), co-injected with 100, 50, 25, or 0 pig of non-radioactive TSHR-Ab, were sacrificed immediately after PET imaging at 18 h p.i. for biodistribution analysis to verify the PET results. Tissues, including blood, liver, kidney, spleen, heart, muscle, bone, and tumors were harvested and weighted, and their radioactivities were measured by a y counter. The data were decay-corrected and expressed as the percentage injected dose per gram of tissue (%ID / g).

[0144] After PET imaging, ex vivo biodistribution experiments for64Cu-NOTA-TSHR-Fab or64Cu-NOTA- TSHR-scFv were conducted in mice bearing TH J529TTSHR+tumors at 24 h p.i. (n = 3). Tissues, including blood, heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, femur, muscle, brain, and tumors were harvested and weighted, and their radioactivity was measured by a gamma counter. The data were decay- corrected and expressed as the percentage injected dose per gram of tissue (%ID / g).Oncocytic thyroid carcinoma cell line and cell culture

[0145] The only characterized human OTC cell line XTC-UC1 expressed TSHR was obtained from Dr. Copland's lab at Mayo Clinic, Jacksonville, Florida, and used for in vitro and in vivo studies. XTC-UC1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fischer Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco) in a humidified incubator at 37°C and 5% CO2. Cells were removed from flasks for passage or transfer to assay plates. TSHR expression in XTC-UC1 cell line was validated and quantified by immunocytochemistry (ICC). Short tandem repeat (STR; aka DNA fingerprinting) was performed on all cell lines by IDEXX BioAnalytics using CellCheckl 6 human Marker STR Profile and Inter-species Contamination Test. Identity matching score above 80% was consistent with cell line of origin.TSHR CAR-T cell therapy in OTC of XCT.UC1 tumor mouse models

[0146] In Vivo Models: Male and female 6-8-week-old NOD-SCI D-l L2ry_ / _(NSG) mice were obtained from Jackson Laboratories (Jackson Laboratories, Bar Harbor, ME, USA) and maintained in an animal barrier space that is approved by the institutional Biosafety Committee for BSL2+ level experiments at the Mayo Clinic Florida animal facility (IBC #HIP00000252.20). Mice were treated on an lACUC-approved protocol (A00001767). In cell line xenograft models, XTC.UC1 cells were suspended in a 50% Matrigel basement medium and subcutaneously injected (2 x 106cells / IOOpil) into the rear flank of NSG mice. Serial caliper measurements were performed to confirm tumor engraftment. Mice were then randomized based on tumor burden to receive different treatments as outlined in the specific experiment. Tumor growth was monitored by serial caliper measurements (width x heightDocket No. 31134 / 2023-456 / PC x length x 0.523 = mm3). Mice were euthanized once lACUC-approved endpoint criteria were met, and remaining tumors were collected for IHC as indicated in the specific experiment. Upon euthanasia, tumors were harvested for IHC. When tumors reached ~100mm3, all mice were randomized based on tumor burden and injected with 10 x 106UTD or TSHR CAR-T as previously described.

[0025] When tumors reached to about 100-300 mm3for PET imaging and biodistribution studies. Disease progression was monitored through serial assessment of tumor volume with caliper measurements. Mice were euthanized at lACUC-approved endpoints.Dosimetry Calculation

[0147] The dosimetry of [64Cu]Cu-NOTA-TSHR-Ab was calculated from the injected dose activity of each animal organs. Specifically, absorbed doses to relevant organs were calculated based on non-decay-corrected ex vivo biodistribution data obtained from healthy non-tumor bearing NSG mice injected with [64Cu]Cu-NOTA- TSHR-Ab. Mice were randomly divided into two groups. Group 1 received 2-3 MBq (100-200 piL) of [64Cu]Cu- NOTA-TSHR-Ab co-injected with 25 pig of non-radioactive TSHR-Ab via the tail vein, while group 2 received the same dose of [64Cu]Cu-NOTA-TSHR-Ab alone. At 2.5, 24, and 48 h post-injection (n = 3 per time point), mice were sacrificed and the mass and radioactivity of selected organs and tissues were measured. Residence times were calculated for selected organs using time-activity curves and mouse-to-human organ scaling factors. Human dosimetry estimates were then generated using the OLINDA / EXM software.TSHR-targeted Radioimmunotherapy for treating thyroid cancer

[0148] To evaluate the potential therapeutic capacity of67Cu-NOTA-TSHR-Ab, THJ529TTSHR+tumor-bearing mice were randomly divided into five groups (n = 5): high dose (0.4 mCi), medium dose (0.2 mCi), low dose (0.1 mCi), TSHR-Ab (25 pig), and saline control. Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded one to three times per week. Tumor volumes were calculated from serial caliper measurements using the formula: tumor = height x length x width x 0.5236.

[0149] To evaluate the potential therapeutic capacity of177Lu-DOTA-TSHR-Ab, THJ529TTSHR+tumor-bearing mice were randomly divided into five groups: high dose (0.3 mCi, n = 5), medium dose (0.2 mCi, n = 5), low dose (0.1 mCi, n = 5), TSHR-Ab (25 pig, n = 3), and saline control (n = 3). Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded every three days. Tumor volumes were calculated from serial caliper measurements using the formula: tumor = height x length x width x 0.5236.

[0150] To evaluate the potential therapeutic capacity of225Ac-DOTA-TSHR-Ab, THJ529TTSHR+tumor-bearing mice were randomly divided into four groups: high dose (20 kBq, n = 5), medium dose (10 kBq, n = 5), low dose (5 kBq, n = 5), and saline control (n = 3). Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded every three days. Tumor volumes were calculated from serial caliper measurements using the formula: tumor = height x length x width x 0.5236.Docket No. 31134 / 2023-456 / PCStatistical analysis

[0151] All numerical data are shown as mean ± S.D. Statistical analysis was performed using GraphPad Prism version 10.1. Data were analyzed using the unpaired, two-tailed Student's t-test and one-way ANOVA. Differences at the 95% confidence level (P < 0.05) were considered statistically significant.Example 1 : TSHR-antibody bioconjugation and radiolabeling with Zr-89 (Construct A1)

[0152] A human monoclonal autoantibody K1-70 was selected for TSHR-targeted radiotracer development due to a high binding affinity, TSHR antagonist activity blocking TSHR mediated cell proliferation, and clinical safety and efficacy profile. The schematic representation of TSHR antibody K1-70 bioconjugation with chelator and radiolabeling with Zr-89 is shown in FIG. 1A. The average number of DFO chelators per antibody K1-70 molecule was determined to be 2.0 by the isotope dilution method

[0028] ,89Zr-DFO-TSHR-Ab was prepared in radiochemical yields of 68.8 ± 9.9% (n = 5) at end of synthesis based on the starting89Zr oxalate radioactivity. The radiochemical purity of this radiotracer after PD-10 separation was 98.7 ± 0.8% as determined by radio-TLC (FIG. 1B). The specific activities of this radiotracer were 19.1 ± 2.7 mCi / mg for further in vitro and in vivo evaluation.Example 2: In Vitro evaluation of89Zr-DFO-TSHR-Ab (Construct A1)

[0153] Radiotracer89Zr-DFO-TSHR-Ab remained stable (> 95%) in formulation solution at room temperature for 3 days based on radiochemical purity analysis by radio-TLC. Further in vitro evaluation of89Zr-DFO-TSHR-Ab cell uptake assay used three TSHR-expressing cell lines TSHR-K562, TSHR-THJ-529T, and TSHR-FTC-133 as positive controls and their WT cell lines K562, THJ-529T, and FTC-133 as negative controls (FIG. 2A). TSHR expression level was analyzed by immunocytochemistry in WT cells and cell lines with stably expressed TSHR. WT cells showed no expression of TSHR protein. All three cell lines, TSHR-THJ-529T, TSHR-FTC-133 and TSHR-K562, presented high TSHR expression (dark staining). Cell uptake experiments demonstrated higher specific uptake of89Zr-DFO-TSHR-Ab in all three TSHR-expressing cell lines compared to their corresponding WT. These high uptake levels could be blocked in the presence of unlabeled TSHR antibody K1-70 (20 pig). (FIG. 2B). In addition, both TSHR-K562 and TSHR-THJ-529T demonstrated more89Zr-DFO-TSHR-Ab uptake compared to that of TSHR-FTC-133. Based on the radiotracer cell uptake study, all three TSHR-expressing cell lines were further analyzed for saturation binding affinity expressed as a Kd value and competition radioligand binding assay was expressed as an I C50 value. The89Zr-DFO-TSHR-Ab binding affinity was measured in a saturation binding assay by incubating TSHR-expressing cells with increasing radioligand concentrations. The dissociation constant values (Kd) were 0.58 nM for TSHR-K562 cells (FIG. 2C), 2.31 nM for TSHR-THJ-529T cells, and 3.67 nM for TSHR-FTC-133 cells (FIG. 6E). Furthermore, a competition radioligand binding assay was developed for89Zr-DFO-TSHR-Ab, in the presence of varying concentrations of competing TSHR antibody K1- 70. In this assay, the IC50 values for TSHR antibody K1-70 on TSHR-K562, TSHR-THJ-529T, and TSHR-FTC- 133 cell line were 0.81 nM (FIG. 2D), 1.04 nM, and 0.84 nM (FIG. 6F), respectively. Collectively, these in vitroDocket No. 31134 / 2023-456 / PC cell data demonstrated that89Zr-DFO-TSHR-Ab is highly specific for TSHR-expressing cells with high potency, and suitable for further in vivo evaluation.Example 3: In vivo evaluation of89Zr-DFO-TSHR-Ab (Construct A1)

[0154] Immuno-PET imaging of89Zr-DFO-TSHR-Ab was first examined in a subcutaneous tumor NSG mouse model (n= 3), whose left flank was implanted with WT K562 tumor (TSHR negative), and right flank was implanted with TSHR-K562 tumor (TSHR positive). The 24, 48, and 72 h post-injection (p.i.) imaging time points were chosen for serial PET scans after intravenous89Zr-DFO-TSHR-Ab injection with or without TSHR antibody K1-70 (100 pg). Representative coronal projection images of89Zr-DFO-TSHR-Ab PET-CT after injection of 50 piCi of89Zr-DFO-TSHR-Ab in a subcutaneous mouse model with TSHR negative K652 tumor in the left flank (left facing arrow) and TSHR positive tumor TSHR-K652 in the right flank (right facing arrow) from 24 hours to 72 hours was shown in FIG. 3A. The PET and CT images are presented in FIG. 7A and 7B, (with and without cold K1-70 antibody coinjection, respectively). The quantitative data obtained from ROI imaging analysis were shown in FIG. 3B. The PET imaging and the SUV quantification data demonstrated significant differences in the uptake of89Zr-DFO-TSHR-Ab in TSHR-positive tumors (average SUVmax 1.6 ± 0.2, 1.3 ± 0.1, and 1.2 ± 0.1) versus TSHR-negative tumors (SUVmax 0.8 ± 0.2, 0.7 ± 0.2, and 0.6 ± 0.1) at all three imaging time points. TSHR- positive tumors were about two times higher than TSHR-negative tumor uptakes. These different uptakes of89Zr- DFO-TSHR-Ab in TSHR positive and negative tumors demonstrated that this radiotracer is specific to PET imaging of TSHR expression. Besides tumor uptake,89Zr-DFO-TSHR-Ab PET demonstrated expected antibody distribution including high uptake in the liver, and lower uptake in muscle. Collectively, the whole-body PET / CT imaging of89Zr-DFO-TSHR-Ab in tumor xenograft mice demonstrate that this radiotracer has the potential for PET imaging TSHR expression with reasonable tumor uptake and contrast.

[0155] To further demonstrate the specificity of89Zr-DFO-TSHR-Ab for PET imaging of TSHR expression, a blocking study using the coinjection of the radiotracer with 100 pg of TSHR antibody K1-70 was performed in the same mouse model (n =3). The representative PET / CT fused images from the block group were shown in FIG. 3C. The PET and CT images were presented in FIG. 7. The quantitative data of89Zr-DFO-TSHR-Ab with TSHR antibody coinjection obtained from ROI imaging analysis are shown in FIG. 3D. Similar to the group without the coinjection of TSHR antibody, the PET imaging and the SUV quantification data demonstrated a significant difference in the uptake of89Zr-DFO-TSHR-Ab in TSHR positive tumors (SUVmax 2.8 ± 0.1, 2.7 ± 0.4, and 2.9 ± 0.1) compared to that of TSHR negative tumors (SUVmax 1.3 ± 0.2, 1.1 ± 0.1, and 1.2 ± 0.0) at 24 h, 48 h, and 72 h time points, respectively. Co-injection with TSHR antibody K1-70 (100 pg) significantly enhanced both tumor uptakes, which showed that TSHR-expressing tumors had the highest radioactive uptake and were 2.2 to 2.4 times higher than these uptakes of TSHR-negative tumors, believed to be due to blocking low levels of nonspecific89Zr-DFO-TSHR-Ab binding in physiologic tissues. Specifically,89Zr-DFO-TSHR-Ab coinjection PET showed higher initial blood radioactivity accumulation, but lowered other major organ89Zr-DFO-TSHR-Ab uptake including liver and kidney. This blocking study demonstrated the coinjection of TSHR antibody K1-70 (100 pg) enhanced this radiotracer for PET imaging of TSHR expression, and the whole-body PET / CT imaging of89Zr-Docket No. 31134 / 2023-456 / PCDFO-TSHR-Ab with low specificity or TSHR antibody coinjection can specifically image TSHR-positive tumor with more favorable imaging characteristics (higher tumor uptake and contrast) compared the group without the antibody coinjection.

[0156] The tumor PET imaging pharmacokinetic data was further compared. FIG. 4A showed the time-activity curves of all tumors after the injection of89Zr-DFO-TSHR-Ab with / without the antibody coinjection. In the group without TSHR antibody coinjection, both TSHR positive and negative tumor uptake demonstrated similar pharmacokinetic pattern, which TSHR-negative tumor uptake slightly decreased from average SUVmax 0.8 to 0.6 during the imaging time from 24 to 72 hours and corresponding TSHR-positive tumor uptake moderately dropped from 1 .6 to 1 .2. With the decrease of tumor uptake overtime from 24 hours to 72 hours, the imaging contrast, expressed as SUV ratio of TSHR-expressing tumor to organ, such as the ratio of tumor to liver and the ratio of tumor to blood remained stable at 0.6 ± 0.1 and 5.4 ± 1 .5, respectively, but the ratio of tumor to muscle showed significantly decreased levels from 8.0 ± 1.0 to 2.7 ± 0.9 (FIG. 4B). In the TSHR antibody coinjection group, both TSHR positive and negative tumor uptake demonstrated similar pharmacokinetic patterns but difference from the group without coinjection. Specifically, radioactive uptakes of both TSHR negative and positive tumors remained stable during the imaging time from 24 to 72 hours, and the average SUVmax values of TSHR negative and positive tumors were changed from 1.3 to 1.2 and from 2.8 to 2.9, respectively. The TSHR- positive tumor uptake was 2.2-2.4 times higher than TSHR-negative tumor uptake within TSHR antibody coinjection group. The tumor imaging contrast (2.2-2.4) is higher than the group (2.0) without TSHR antibody coinjection. The imaging contrast of tumor to non-target organs in the antibody coinjection group from 24 hours to 72 hours, also presented a more favorable pattern with the change. The ratios of tumor to liver, muscle, and blood were increased from 1.8 ± 0.2, 8.4 ± 1.3, 1.7 ± 0.1, and 1.7 ± 0.1 to 2.1 ± 0.5, 8.7 ± 1.7, and 3.1 ± 0.6, respectively. (FIG. 4C).

[0157] To match antibody K1-70 biological half-life, Zr-89 was chosen for this full-length human monoclonal antibody K1-70 radiolabeling as a proof-of-concept study. Although the radiotracer89Zr-DFO-TSHR-Ab PET study demonstrated specific uptake of TSHR-expressing tumors, it reached at an early time point (24 hours) with moderate accumulation and washed out gradually. This observation is consistent with a recent report of TSHR- targeted PET imaging with the agonist-based radiotracer89Zr-TR1402

[0018] , Besides tumor uptake, liver, kidney, muscle, and blood SUV values were analyzed (FIG. 3A) and results were verified with follow-up biodistribution analysis (FIGS. 5A-5C) to compare the radiotracer clearance in this study. To further investigate the specificity of this radiotracer, a blocking study was performed using the coinjection of the radiotracer with cold unlabeled TSHR antibody K1-70 (100 pg) in excess. Surprisingly, the 100 pg antibody coinjection did not block TSHR positive tumor uptake, and indeed enhanced both TSHR positive and negative tumor uptake of the89Zr-DFO- TSHR-Ab, especially TSHR-expressing tumor by more than 2-fold as well as significantly lowered clearance organs like liver and kidney uptake (FIG. 3B). Without wishing to be bound by any particular theory, it is believed that TSHR is shed in the blood and disturbed by endogenous TSH binding, or "TSHR antigen sink” effect, which TSHR is also expressed in non-tumor organs or tissues, such as bone marrow or spleen, and the antibody K1-70Docket No. 31134 / 2023-456 / PC coinjection could be beneficial to improve the tumor uptake, because this low molar activity or carrier-added radiotracer would suppress non-specific radiotracer binding in antigen-expressing normal tissues while increasing uptake within the tumor. This effect of carrier-added radiotracer increasing tumor uptake has also been observed in other antibody radiotracers in preclinical and clinical studies [31, 32], Indeed, a noticeable high uptake in both bone and spleen was observed, similar to the levels of liver uptake in the group without the antibody coinjection. The high bone and spleen uptake significantly dropped in the group with the antibody coinjection. Without wishing to be bound by any particular theory, this difference may contribute to the involvement of TSHR expression in murine dendritic cells of spleen and bone marrow macrophages [33, 34], further supporting the hypothesis.

[0158] TSHR antibody coinjection significantly increased the TSHR-expressing tumor uptake and the imaging contrast to other organs (FIG. 3C and FIG. 3D). However, in vivo pharmacokinetics of this antibody-carried radiotracer only showed moderately increased tumor uptake in a later 72-hour time point. Furthermore, this antibody-carried radiotracer cleared from blood circulation was noticeably slow, as shown by higher blood uptake, which indicated that imaging time-points could be extended to later time points, such as day 5 or day 7, for achieving higher tumor uptake and imaging contrast. Nevertheless, this outstanding higher uptake and retention of TSHR-expressing tumor and lower uptakes in other organs provide preliminary data for further clinical translation of this radiotracer in the first-in-human study, and further investigate the potential of TSHR as a target for radiopharmaceutical therapy development.Example 4: Ex vivo evaluation of89Zr-DFO-TSHR-Ab (Construct A1)

[0159] After the PET / CT imaging of89Zr-DFO-TSHR-Ab at 72 h post-injection, all mice were sacrificed for ex vivo biodistribution study to quantitatively verify the PET imaging data (FIG. 5A). The biodistribution data of89Zr- DFO-TSHR-Ab at 72 h post-injection were consistent with the results of PET study. The biodistribution data showed a significant difference in radioactive uptake of tumors, expressed in %ID / g, in the presence and absence of TSHR antibody K1-70 (100 pg). Briefly, the average radioactive uptake in TSHR-negative tumor without or with the coinjection of TSHR antibody was 3.89% and 8.98% I D / g, respectively. The corresponding TSHR-positive tumor without or with the coinjection of TSHR antibody had 3.94% and 11 .72 %ID / g, respectively, which both were higher than the TSHR-negative tumor uptake, shown in FIG. 5B. Furthermore, both TSHR positive and negative tumor tissues were sampled for immunohistochemical staining. The data derived from biodistribution and PET imaging of TSHR positive tumors agreed with that of TSHR IHC of tumor tissues. Other major tissues, including blood, liver, kidney, and muscle showed similar radioactive uptakes, and ratios of TSHR positive tumors to these tissues from biodistribution were similar to the data presented in PET imaging analysis. Overall, the quantification results obtained from biodistribution studies and PET scans matched well, confirming that quantitative ROI analysis of noninvasive micro-PET / CT scans accurately reflected the distribution of89Zr- DFO-TSHR-Ab in vivo with or without TSHR antibody coinjection.Docket No. 31134 / 2023-456 / PC

[0160] The data in Examples 1-4 clearly show that TSHR is a promising target for PET imaging of thyroid cancer. Besides the human monoclonal antibody K1-70, a few new biologies including small molecules, peptides, antibody fragments, and antibodies have been reported as under investigation for TSHR-targeted therapy

[0035] , These TSHR antagonists and agonists could serve as lead compounds for both diagnostic and therapeutic radiopharmaceutical development due to their improved nanomolar binding affinities. Current standard of care therapy for metastatic thyroid cancer consists of radioactive iodine which is essentially a radiotheranostic i.e. diagnostic and therapeutic. However, more effective p-emitting radionuclides (e.g., Lu-177 and Cu-67) or o- emitting isotopes (e.g., Ac-225 and Pb-212) provide greater mean tissue range and linear energy transfer for TSHR radiopharmaceutical development, allowing for a much greater radiation dose being delivered to the thyroid tumor and tumor microenvironment

[0036] , The promising tumor uptake and retention, biodistribution, and pharmacokinetics of antibody-carried radiotracer generated from this study pave the way for the development of TSHR-targeted radiotheranostics for thyroid cancer imaging and therapy.Example 5: Evaluation of TSHR expression in PTC XTC-UC1 tumor bearing miceTSHR expression in normal thyroid and OTC XTC-UC1 tumor

[0161] TSHR expression levels were analyzed using a thyroid cancer biobank and XTC-UC1 tumor grown in a mouse model. The analysis demonstrated that TSHR is highly expressed on benign and malignant thyroids.

[0058] Immunohistochemical (IHC) staining confirmed TSHR expression on XTC-UC1 tumor, which demonstrated XTC- UC1 tumor presented higher TSHR expression (FIG. 8B, dark staining) similar to normal thyroid tissue (FIG. 8A). FIG. 8C depicts a graph of the TSHR expression level quantified using H-Score (0-3 intensity scoring) and the percentage of total areas corresponding to each score.**** p <0.0001 , unpaired f-test.PET / CT imaging of TSHR expression in OTC XTC-UC1 tumor bearing mice

[0162] PET / CT imaging of89Zr-DFO-TSHR-Ab was performed in the clinically relevant OTC mouse model UCT.UC1 . Representative PET / CT images obtained at 24, 72 and 120 h post-injection of89Zr-DFO-TSHR-Ab are shown in FIGS. 9A and 9B. In another group, the mice were treated with TSHR CAR-T therapy, and 3 days later, the mice were rechallenged with XTC-UC1 cells implantation (left rear flank, right-pointing arrow) (FIG. 9B). FIG. 9B demonstrated lower radiotracer accumulation after TSHR-CART therapy from 24 hours (SUV=0.4) to 120 hours (SUV=0.3) and inhibited OTC growth and attenuated TSHR expression as indicated by the right-pointing arrow where newly injected XTC.UC1 (left flank) and the right flank of the original injected cells (left-pointing arrow).

[0163] PET / CT imaging of89Zr-DFO-TSHR-Ab was performed in the clinically relevant OTC mouse model UCT.UC1 . Representative PET / CT images obtained at 24, 72 and 120 h post-injection of89Zr-DFO-TSHR-Ab are shown in FIG. 9C. After tail vein injection of the radiotracer, small-animal PET / CT imaging (24 h, 72 h, and 120 h) were conducted on XTC-UC1 tumor-bearing mice (n = 4) before TSHR CAR-T therapy (Pre-CAR-T). Serial PET imaging showed clear delineation of the OTC tumor after 24 hours (SUVmax = 1.19 ± 0.27), with typical Zr-89 labeled antibody uptake in normal tissues, and radiotracer retention in OTC through 120 hours (SUVmax = 0.9 ±Docket No. 31134 / 2023-456 / PC0.16), as shown in FIG. 9C and quantified in FIGs. 9D-9F. In a longitudinal design (n = 3), the same mice were treated with TSHR CAR-T cells and later rechallenged with XTC.UC1 cells after 8 weeks, to evaluate both the durability of the therapeutic response and the development of immune memory. This within-subject design reduces biological variability and allows for more accurate assessment of long-term efficacy. In the Post-CAR-T phase, PET images demonstrated significant reduction in radiotracer accumulation from 24 hours (SUVmax = 0.55 ± 0.1 ) to 120 hours (SUVmax = 0.4 ± 0.08 ), as indicated in FIG. 9C (middle row, arrow) and FIGs. 9D-9F. Upon rechallenge, the same mice developed tumor with slow growth and consistently low tracer uptake, with SUVmax values of 0.52 ± 0.02 at 24 h p.i. and 0.49 ± 0.06 at 120 p.i. (FIG. 9C, bottom row, arrow; FIGs. 9D-9F), strongly supporting the presence of functional immune memory. Therefore, these results demonstrate that89Zr-DFO- TSHR-Ab enables effective noninvasive imaging of TSHR expression in OTC. The sustained reduction in radiotracer uptake following CAR-T therapy and tumor rechallenge in the same animals suggest the durable antitumor efficacy and the potential establishment of TSHR-specific immune memory.Ex vivo analysis of TSHR expression in OTC

[0164] Whole-body89Zr-DFO-TSHR-Ab micro-PET / CT imaging results were further validated by ex vivo biodistribution of radiotracer and IHC assays on TSHR expression of OTC tumor tissue. Following the micro- PET / CT imaging at 120 hours post-injection (p.i.) in the Pre-CAR-T group, all mice were sacrificed for biodistribution studies. In addition, a separate group of three mice were injected with89Zr-DFO-TSHR-Ab and euthanized at 24 hours p.i. for early time point biodistribution studies. The biodistribution data of89Zr-DFO-TSHR- Ab at 24 h and 120 hours p.i. expressed in %ID / g, were shown in FIG. 10A. Briefly, the biodistribution data including blood, liver, kidney, muscle, and tumor, at 24 hours and 120 hours p.i showed a similar pattern in radioactive uptake, expressed in %ID / g. The uptakes of89Zr-DFO-TSHR-Ab in all selected organs or tissues at 24 hours p.i were significantly higher than these uptakes at 120 hours p.i due to the washout. Radiotracer accumulation in OTC tumors were 5.51 ± 1.34 %ID / g, and 3.42 ± 0.46 %ID / g at 24 hours, and 120 hours p.i, respectively, indicating sustained retention of the radiotracer in TSHR-positive tumors. Notably, tumor uptake was significantly higher than in blood and muscle, but lower than in the liver, which is consistent with hepatic clearance and Fc receptor-mediated uptake. Overall, the quantification results obtained from ex vivo biodistribution studies and in vivo mico-PET / CT imaging were consistent, demonstrated that quantitative ROI analysis of micro-PET / CT scans accurately reflected the distribution of89Zr-DFO-TSHR-Ab in mice bearing OTC tumor. TSHR expression on OTC tumor tissue was confirmed by the IHC (FIG. 10B). Collectively,89Zr-DFO- TSHR-Ab PET could noninvasively and quantitatively detect TSHR-expression in OTC.

[0165] The surface glycoprotein receptor, TSHR is a major thyroid autoantigen and plays a key role in the regulation of thyroid function and metabolism. Accumulating studies showed high TSHR expression (i.e. no loss of expression) in thyroid cancers including OTC. As noted previously, Gimblet et al. demonstrated that 62% of OTC patient tumors retain expression of TSHR

[0046] while Shih et al. showed 150 / 197 (76%) of OTC patient tumors overexpress TSHR.

[0064] TSHR expression was measured in tissues obtained from patients with OTC, and it was found that 100% of tumors retained TSHR expression; however, recurrent and metastatic lesionsDocket No. 31134 / 2023-456 / PC levels were attenuated -60% (unpublished data). However, most known thyroid cancer cell lines lose expression of TSHR in two-dimensional (2D) cell culture with the exception of one cell line, XCT.UC1 . In this present study, using XCT.UC1 cells,89Zr-DFO-TSHR-Ab PET imaging for noninvasive assessment of TSHR expression in OTC in mice bearing XCT.UC1 tumor has been conclusively demonstrated.

[0166] Additionally, potent antitumor activity of TSHR targeted CAR-T therapy against the XCT.UC1 mouse model has recently been reported.

[0058] Directed TSHR PET / CT imaging with89Zr-DFO-TSHR-Ab in XCT.UC1 bearing tumors undergoing CAR-T therapy has the potential to serve as an imaging biomarker and also allows for longitudinal and quantitative assessment of precision TSHR CAR-T therapy, and can function as a companion diagnostic test for selecting patients who will benefit from this novel TSHR targeted CAR-T immunotherapy. To achieve this goal, repeated micro-PET / CT imaging was performed of89Zr-DFO-TSHR-Ab on mice bearing XCT.UC1 tumors before and after TSHR CAR-T therapy.89Zr-DFO-TSHR-Ab tumor uptake significantly dropped in responding XCT.UC1 tumors after TSHR CAR-T therapy and XCT.UC1 tumors, newly implanted after CAR-T, did not demonstrate uptake of89Zr-DFO-TSHR-Ab, consistent with successful targeted therapy against the TSHR expression. Without wishing to be bound by any particular theory, it is thought that this finding implies that TSHR PET / CT with89Zr-DFO-TSHR-Ab can prognosticate which OTC tumors will respond to CAR-T therapy and can also be used to monitor response to TSHR CAR-T cell therapy. Additionally, the lack of89Zr-DFO-TSHR-Ab uptake in newly implanted OTC tumors after 8 weeks of TSHR CAR-T cell therapy, there persists activation of CAR-T protecting against OTC recurrent disease.

[0167] The selective TSHR antibody K1-70 was labeled using zirconi um-89 (89Zr), a positron-emitting radionuclide with a half-life of 78.4 hours with the intention of radioactive half-life matching that of the circulating half-life of the antibody. However, the data presented herein show that89Zr-DFO-TSHR-Ab PET can detect TSHR expression as early as 24 hours with reasonable uptake and high imaging contrast (FIG. 9A). Therefore, it may be clinically favorable to develop radiolabeled TSHR antibody K1-70 or its fragment using a short half-life positron emitter, such as copper-64 with a half-life of 12.7 hours, and Ga-68 (t1 / 2 =68 minutes). In addition,89Zr- DFO-TSHR-Ab has moderate SUV value in the OTC XCT.UC1 tumor. Previous studies have found that coinjection TSHR antibody with radiotracer could increase tumor uptake more than two times and remains the uptake retention.

[0031] With increased tumor uptake and retention, the successful development of89Zr-DFO-TSHR- Ab for PET imaging of TSHR expression for OTC could serve as a template for other radiometal-based theragnostic agent development by changing imaging positron emitter with therapeutic radionuclide. For instance, replacing Zr-89 or Cu-64 with other radionuclides like Cu-67, Lu-177, or Ac-225 to label TSHR-Ab or its small-size equivalent molecules conjugating with other chelator, resulting in novel theragnostic agents for OTC tumor radioligand therapy. [65, 66]

[0168] This study demonstrates that89Zr-DFO-TSHR-Ab PET imaging clearly delineated OTC tumor with reasonable uptake and high contrast and monitored TSHR CAR-T therapy in the xenograph model of human tumor. The results suggest that89Zr-DFO-TSHR-Ab is a potentially reliable biomarker to identify OTC tumors and predict TSHR CAR-T therapy response in patients with invasive and metastatic OTC.Docket No. 31134 / 2023-456 / PCExample 6: Synthesis of64Cu-NOTA-TSHR-Ab (Construct A2)

[0169] 64Cu-NOTA-TSHR-Ab was prepared by complexing64CuCl2 with the conjugate NOTA-TSHR-Ab, shown in FIG. 11 A and described below. Briefly,64CuCl2 (10-20 mCi in 10-20 piL of 0.05 M HCI aqueous solution) was mixed with 0.1 M ammonium acetate buffer (pH 5.5, 200 pL) to pH 5.3-5.5 at room temperature. The conjugate NOTA-TSHR-Ab (80-100 pig) (characterized by HPLC shown in FIG. 11C) in 1 M HEPES buffer (pH 7.4) solution (30 piL) was added to the above64Cu-solution and incubated at 37 °C for 60 minutes for radiolabeling. The resulting64Cu-NOTA-TSHR-Ab mixture was purified using a PD10 gel filtration column with PBS (pH=7.4) or normal saline solution as eluent. The radiochemical identity and purity of the final product64Cu- NOTA-TSHR-Ab were determined by radio-HPLC (FIG. 11 D) and radio-TLC with 50 mM EDTA (pH 8.0) as the developing solution, shown in FIGs. 11 B and 11 E.Example 7: In vitro evaluation of64Cu-NOTA-TSHR-Ab (Construct A2)

[0170] To assess the targeting potential of64Cu-NOTA-TSHR-Ab for TSHR-positive thyroid tumor cells, a series of in vitro uptake and binding affinity studies was conducted using THJ529TreHR+or THJ529T14'7' cells. The uptake of64Cu-NOTA-TSHR-Ab was significantly higher in THJ529TreHR+cells (%uptake / mg: 33.85 ± 4.71) compared to TH J529T14''7’ cells negative for TSHR expression (%uptake / mg: 4.13 ± 1.11, P < 0.0001). This uptake was substantially reduced by the addition of an excess of non-radioactive TSHR-Ab (%uptake / mg: 16.58 ± 0.70, P < 0.001), indicating the high cellular specificity of64Cu-NOTA-TSHR-Ab binding to TSHR-positive thyroid tumor cells (FIG. 12A). It was also observed that the uptake in TH J529TreHR+cells would increase with incubation time extension (FIG. 12D), which become 34.53 ± 0.78 %uptake / mg at 1 h and reached 86.70 ± 4.37 %uptake / mg after a 3-h incubation. Furthermore,64Cu-NOTA-TSHR-Ab cell studies demonstrated a nanomolar binding affinity for THJ529TTSHfi+cells, with a Kdof 4.74 nM (FIG. 12B) and a Ki of 0.92 nM (FIG. 12C).Immunoreactivity assay of [64Cu]Cu-NOTA-TSHR-Ab was also conducted which exhibited high immunoreactivity with a rvalue of 0.87 (FIG. 12E). These in vitro results revealed the good specificity and affinity of64Cu-NOTA- TSHR-Ab binding to TSHR-positive thyroid tumor cells, supporting its diagnostic potential in subsequent in vivo experiments.Example 8: In vivo evaluation of64Cu-NOTA-TSHR-Ab (Construct A2)

[0171] Immuno-PET imaging of64Cu-NOTA-TSHR-Ab was examined in a subcutaneous tumor NSG mouse model (n= 6, 3 male and 3 female), whose left flank was implanted with THJ-529T WT tumor (TSHR negative), and right flank was implanted with THJ-529T-TSHR tumor (TSHR positive). The 1, 2, 4, 18, 24, and 48 h postinjection (p.i.) imaging time points were chosen for serial PET scans after intravenous64Cu-NOTA-TSHR-Ab injection with TSHR antibody K1-70 (100 pg). Representative coronal projection images of64Cu-NOTA-TSHR-Ab PET-CT after injection of 150 piCi of radiotracer in a subcutaneous mouse model with TSHR negative THJ-529T WT tumor in the left flank (left facing arrow) and TSHR positive tumor THJ-529T-TSHR in the right flank (right facing arrow) from 4 hours to 48 hours was shown in FIG. 13A. Representative coronal projection images of64Cu-NOTA-TSHR-Ab PET-CT after injection of 150 piCi of radiotracer in a subcutaneous mouse model withDocket No. 31134 / 2023-456 / PCTSHR negative THJ-529T WT tumor in the left flank (left arrow) and TSHR positive tumor THJ-529T-TSHR in the right flank (right arrow) from 1 hours to 48 hours was shown in FIG. 13C. The quantitative tumor uptake data obtained from ROI imaging analysis were shown in FIG. 13B. The PET imaging and the SUV quantification data demonstrated significant differences in the uptake of64Cu-NOTA-TSHR-Ab in TSHR-positive tumors (average SUVmax 15.9, 24.2, 25.5, and 27.3) versus TSHR-negative tumors (average SUVmax 7.1 , 10.9, 12.9 and 18.7) at all four imaging time points. [64Cu]Cu-NOTA-TSHR-Ab PET showed initially high TH J529TTSHR+tumor uptake (SUVmax: 2.00 ± 0.65 at 2 h p.i .), which continued to increase over time (SUVmax: 2.38 ± 0.84, 3.63 ± 0.42, 3.82 ± 0.44, and 4.09 ± 0.56 at 4, 18, 24, and 48 h p.i., respectively). Notably, [64Cu]Cu-NOTA-TSHR-Ab uptake was observed in both THJ529TTSHR+and THJ529TWTtumors; however, uptake in TH J529TTSHR+tumor was consistently higher than in THJ529TWTtumor, with the difference being most pronounced at 18 h (T / NT: 2.23 ± 0.27) and 24 h (T / NT: 1.98 ± 0.25) p.i. TSHR-positive tumors were about two times higher than TSHR-negative tumor uptakes from 4 to 24 hours. These different uptakes of64Cu-NOTA-TSHR-Ab in TSHR positive and negative tumors demonstrated that this radiotracer is specific to PET imaging of TSHR expression. Besides tumor uptake,64Cu-NOTA-TSHR-Ab PET demonstrated expected antibody distribution including low uptake in the liver, kidney, heart, muscle, bone, and brain. Background signals from organs such as the blood (SUVmax: 3.68-4.57), liver (SUVmax: 2.32-2.65), and kidney (SUVmax: 2.25-2.55) were relatively high during the initial 4 h but showed a decline from 18 to 48 h p.i. (SUVmax: 1.59-2.35 for blood, 1.80-2.01 for liver, and 1.44-1.63 for kidney) (FIG. 13D). Consistently low uptake in bone (SUVmax: 0.5-1.0) and muscle (SUVmax: <0.5) was also expected. Additionally, T / NT ratios of the liver, blood, bone, kidney, and muscle all showed an increasing trend over time, indicating that imaging in a later time point (18 or 24 h p.i.) could achieve better image contrast (FIG. 13E). Although increasing uptake in TH J529T14''7’ tumors was observed, TSHR IHC analysis of the tumor following PET imaging demonstrated strong TSHR expression in THJ529TreHR+tumors (brown staining) and low TSHR expression in THJ529T14'7' tumors (FIG. 13F). Collectively, the whole-body PET / CT imaging of64Cu-NOTA-TSHR- Ab in tumor xenograft mice demonstrate that this radiotracer has the potential for PET imaging TSHR expression with reasonable tumor uptake and contrast.

[0172] To further optimize the effect of non-radioactive TSHR-Ab dosage on PET / CT imaging, THJ529TreHR+tumor-bearing mice were co-injected with 100, 50, 25, or 0 pig of non-radioactive TSHR-Ab, followed by PET / CT imaging at 18 h p.i. (FIG. 14A). PET images and corresponding quantitative analysis revealed that co-injection of non-radioactive TSHR-Ab significantly reduced background uptake in the spleen, liver, and bone, while increasing blood circulation time (FIG. 14A-14C). The reduction in background uptake improved images contrast, but the effect on tumor and background varied depending on the non-radioactive TSHR-Ab dose. In this study, mice without non-radioactive TSHR-Ab coinjection exhibited the SUVmax of 6.11 ± 1 .07 for tumor, 3.52 ± 0.26 for liver, 3.56 ± 1.01 for bone, and 0.54 ± 0.10 for blood. After co-injection of non-radioactive TSHR-Ab, 25 pig of non-radioactive TSHR-Ab significantly reduced background signal without affecting tumor uptake (SUVmax: 6.31 ± 0.56 for tumor, 2.22 ± 0.08 for liver, 0.82 ± 0.1 1 for bone, 1.70 ± 0.19 for blood), higher doses of 50 pig (SUVmax: 4.35 ± 0.08 for tumor, 2.09 ± 0.02 for liver, 0.64 ± 0.15 for bone, and 2.07 ± 0.26 for blood) and 100 pigDocket No. 31134 / 2023-456 / PC(SUVmax: 4.37 ± 0.22 for tumor, 2.02 ± 0.25 for tumor, 0.55 ± 0.08 for bone, and 2.38 ± 0.12 for blood) led to a slightly attenuation of tumor uptake (FIG. 14B-14C), suggesting partial competitive inhibition. These findings indicate that low doses (25 pig) of non-radioactive TSHR-Ab coinjection effectively reduced background signals without compromising tumor uptake, whereas higher doses (>50 pig) may competitively block tumor uptake. Additionally, non-radioactive TSHR-Ab coinjection showed a slight increase in kidney and muscle radioactivity, which should not be ignored. When determining the optimal dose of non-radioactive TSHR-Ab, it is crucial to balance tumor uptake, blood circulation, and major organ background signals to optimize imaging contrast.Example 9: Ex vivo evaluation of64Cu-NOTA-TSHR-Ab (Construct A2)

[0173] After the PET / CT imaging of64Cu-NOTA-TSHR-Ab at 48 h post-injection, all mice (e.g., THJ529TrsHR+ / M / rand TH J529TreHR+tumor-bearing mice) were sacrificed for ex vivo biodistribution study to quantitatively verify the PET imaging data (FIG. 15A-15B). The biodistribution data of64Cu-NOTA-TSHR-Ab at 18 and 48 h post-injection were consistent with the results of PET study. The biodistribution data (FIG. 15A) revealed high uptake in THJ529TreHRt+ttumor. Despite the absence of TSHR expression, tumor1447' exhibited notable uptake, likely due to the enhanced permeability and retention (EPR) effect or passive diffusion of the antibody. However, uptake in tumor7SHR+remained significantly higher than in tumor14'7, with the most pronounced difference observed at 18 h p.i. (%ID / g: 20.23 ± 2.96 vs. 13.56 ± 1.76, P < 0.0001), highlighting the specificity of64Cu-NOTA-TSHR-Ab. Additionally,64Cu-NOTA-TSHR-Ab uptake in the spleen remained elevated at both 18 h (16.06 ± 4.30%ID / g) and 48 h (17.16 ± 2.37%ID / g), suggesting a strong affinity of TSHR-Ab for spleen- associated immune components. Other major tissues, including blood, liver, kidney, spleen, heart, bone, and muscle showed lower uptakes than tumor (except spleen). Moderate uptake was observed in the blood, liver, kidney, and heart, though these levels decreased significantly by 48 h, indicating systemic clearance over time. Muscle and bone uptake remained consistently low, minimizing non-specific background interference. Further investigation into the effects of non-radioactive TSHR-Ab doses (0-100 pig) demonstrated that co-injection of non-radioactive TSHR-Ab significantly reduced background uptake in the spleen, liver, and bone, without compromising tumor uptake (FIG. 15B). Overall, the quantification results obtained from biodistribution studies and PET scans matched well, confirming that quantitative ROI analysis of noninvasive micro-PET / CT scans accurately reflected the distribution of64Cu-NOTA-TSHR-Ab in vivo with TSHR antibody coinjection. However, SUVmax was used as the metric for quantifying radiotracer accumulation in FIGs.13B and14B and % ID / g was used to indicate the radiotracer uptake in FIGs. 15A-15B. showed radiotracer uptakes in TSHR positive tumors are higher than TSHR negative tumor. Among the tested doses, 25 pig of non-radioactive TSHR-Ab yielded the highest tumor uptake (27.03 ± 2.50 %ID / g), suggesting an optimal balance between tumor targeting and background reduction.Docket No. 31134 / 2023-456 / PCExample 10: Comparison Study of [64Cu]Cu-NOTA-TSHR-Ab and [18F]FDG PET / CT Imaging in A Thyroid Cancer Model

[0174] In this study, the PET / CT imaging performance of a newly developed [64Cu]Cu-NOTA-TSHR-Ab was compared with the widely used radiotracer [18F]FDG in the TSHR-positive THJ529T mouse model, deviated from human poorly differentiated thyroid cancer. PET / CT images and corresponding quantitative analysis revealed a significantly higher tumor uptake of [64Cu]Cu-NOTA-TSHR-Ab compared to [18F]FDG (FIG. 16A-16D). Specifically, [64Cu]Cu-NOTA-TSHR-Ab PET demonstrated a tumor SUVmax of 7.09 ± 0.62, with a tumor-to- muscle ratio of 25.13 ± 9.82, whereas [18F]FDG showed only minimal tumor uptake (SUVmax: 1.51 ± 0.17; T / NT: 3.67 ± 0.61) (FIGs. 16C-16D). These findings indicate the superior specificity of [64Cu]Cu-NOTA-TSHR-Ab for targeting TSHR-positive tumors. In addition to tumor uptake, [64Cu]Cu-NOTA-TSHR-Ab exhibited prominent accumulation in the spleen (SUVmax: 13.44 ± 2.41), bone (SUVmax: 7.50 ± 1.41), and liver (SUVmax: 3.86 ± 0.64) without non-radioactive antibody co-injection (FIG. 16E). Conversely, uptake in the blood (heart, SUVmax: 0.74 ± 0.10) and kidney (SUVmax: 1.34 ± 0.24) was relatively low. In contrast, [18F]FDG exhibited high uptake in the blood (heart, SUVmax: 19.64 ± 3.35) and kidney (SUVmax: 7.70 ± 2.86) but low accumulation in the spleen (SUVmax: 1.13 ± 0.24), bone(SUVmax: 0.76 ± 0.10), and liver (SUVmax: 1.76 ± 0.32). Muscle uptake of both radiotracers remained consistently low (both SUVmax: <0.5), with no significant statistical differences.

[0175] Advanced thyroid cancers are aggressive, lethal, and often resistant to the current standard of care RAI ablation. TSHR expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSHR a compelling target for advanced thyroid cancer imaging and therapy.5 28TSHR expression levels were verified using a thyroid cancer biobank and demonstrated that TSHR is highly expressed on benign and malignant thyroid tissues, including follicular thyroid carcinoma, and papillary thyroid carcinoma based on IHC staining of patient biopsies.29The TSHR-targeting radiotracer [89Zr]Zr-DFO-TSHR-Ab has previously been developed, which presents specific tumor uptake via PET imaging, indicating TSHR as a viable target for novel radiopharmaceutical development. It was observed that [89Zr]Zr-DFO-TSHR-Ab could reach the maximum tumor uptake within 24 h, and the long half-life of zirconium-89, as well as its high energy did not align with in vivo PK / PD of this radiotracer. Copper-64, with a half-life of 12.7 h and low positron energy of 653 keV, may be better suited for TSHR-Ab radiolabeling and imaging, offering much less radiation exposure to patients as a radiodiagnostic, as demonstrated by the FDA-approved PET drug [64Cu]Cu-DOTATATE.30Therefore, a64Cu- labeled selective TSHR antagonist human monoclonal antibody (K1-70) was designed to maintain high tumor uptake and with improved image contrast. K1-70 has demonstrated safety, tolerability, and a lack of immunogenic responses in patients with Graves' disease during a phase I clinical trial (NCT02904330), and a patient with RAI-refractory advanced follicular thyroid cancer and Graves' diseases has successfully been treated using K1-70 under FDA-approved single patient expanded access (compassionate use), demonstrating encouraging therapeutic benefits.31 32Unlike the TSHR agonist RT1401 and TR1402,13 15’33 36immuno-PET with radiolabeled K1-70 offers a novel approach to identify TSHR expression in vivo, as well as providing valuableDocket No. 31134 / 2023-456 / PC diagnostic and therapeutic tools to manage patients with Graves' disease, thyroid cancer, or other thyroid diseases involving TSH-TSHR axis.

[0176] A factor in the success of64Cu-labeled antibody PET imaging is ensuring radiotracer in vivo stability throughout the imaging period, as copper-64 dissociation can lead to unwanted high liver accumulation and compromised image quality. In order to stabilize copper-64 attached to biomolecules, various bifunctional chelators have been investigated, including NOTA,37DOTA,38CB-TE2A,39Sar-cage,40etc. However, DOTA, TETA, and their derivatives for copper-64 labeling have limited in vivo stability due to copper-64 dissociation from these bifunctional chelators, leading to high hepatic uptake and unfavorable hepatobiliary excretion.40Among the available chelators, NOTA is one of the most stable options for copper-64 radiolabeling, and the commercially available p-SCN-Bn-NOTA was selected for this study.1921NOTA-TSHR-Ab was successfully prepared, labeled with copper-64, thereby obtaining [64Cu]Cu-NOTA-TSHR-Ab, which exhibited high stability (RCP > 95% at 48 h; data not shown), high specificity, and nanomolar affinity (Kd = 4.74 nM, Ki = of 0.92 nM; FIGs. 12B-12C) in vitro, making it suitable for further in vivo evaluations. Moderate uptake in liver (SUVmax: 1 .90 ± 0.26, 2.01 ± 0.27, and 1.80 ± 0.22 at 18, 24, and 48 h p.i, FIG. 13D; %ID / g: 11.09 ± 1.04 and 6.44 ± 0.65 at 18 and 48 h, FIG. 15A) was observed, indicating high in vivo stability of [64Cu]Cu-NOTA-TSHR-Ab.

[0177] [64Cu]Cu-NOTA-TSHR-Ab exhibited superior PET imaging performance compared to [89Zr]Zr-DFO- TSHR-Ab. In the presence of 100 pig of non-radioactive TSHR-Ab, [64Cu]Cu-NOTA-TSHR-Ab PET / CT imaging showed earlier and higher uptake in TSHR-positive tumor (SUVmax: 3.63 ± 0.42, 3.82 ± 0.44, and 4.09 ± 0.56 at 18, 24, and 48 h p.i., respectively; FIGs. 13C-13D). In comparison, the previously-developed [89Zr]Zr-DFO- TSHR-Ab displayed lower tumor uptake and a delayed imaging profile (SUVmax: 2.8 ± 0.1, 2.7 ± 0.4, and 2.9 ± 0.1 at 24, 48, and 72 h p.i., respectively).16The higher tumor uptake and prolonged tumor retention of [64Cu]Cu- NOTA-TSHR-Ab highlight its potential as a theranostic agent. Like [89Zr]Zr-DFO-TSHR-Ab, [64Cu]Cu-NOTA- TSHR-Ab exhibited a comparable pharmacokinetic pattern in TH J529T1477tumor and THJ529T7SHR+tumors. However, post-imaging IHC analysis revealed strong TSHR expression in tumor7SHR+and low expression in tumor1477, which did not fully align with PET imaging results. Despite the low TSHR expression, visible radioactivity was observed in tumor1477, although the uptake remained consistently lower than that in the THJ529T7SHR+tumor (data not shown). This discrepancy is likely attributable to the EPR effect, which facilitates passive accumulation of macromolecules in tumors due to high vascular density, large endothelial gaps, and selective extravasation and retention within tumor tissues.41 42Additionally, blood uptake was initially high but gradually declined over time, from an SUVmax of 4.57 ± 1.31 at 1 h to 1.59 ± 0.19 at 48 h p.i., as expected. This clearance pattern suggests that imaging at later time points (after 18 h p.i.) provides optimal image contrast. Therefore, subsequent single-time-point imaging was performed at 18 h p.i.

[0178] An appropriate dose of non-radioactive antibody coinjection can effectively reduce background uptake and enhance image contrast. In a previous study, co-injection of 100 pig of non-radioactive TSHR-Ab (K1-70) with [89Zr]Zr-DFO-TSHR-Ab failed to reduce tumor uptake. On the contrary, it enhanced tumor uptake while reducing background signal. It was hypothesized that this may be due to shed TSHR in the circulation andDocket No. 31134 / 2023-456 / PC competition from endogenous TSH— a "TSHR antigen sink” effect.16Increasing the dose to 200 g did not block tumor uptake either. To further investigate the influence of non-radioactive TSHR-Ab on imaging performance, the non-radioactive TSHR-Ab co-injected in mice with varying doses (100, 50, 25, or 0 pg). These results showed a significant reduction in background uptake (e.g., spleen, liver, and bone), which markedly improved image contrast (FIGS. 14A-14C and FIG. 15B) and may facilitate the detection of small lesions and metastases (e.g., bone metastases). Among the tested doses, 25 pg was identified as optimal, achieving both a significant decrease in background signal and the highest tumor uptake (SUVmax: 6.31 ± 0.56; %ID / g: 27.03 ± 2.50). In contrast, 50 pg (SUVmax: 4.35 ± 0.08; %ID / g: 22.68 ± 8.21) and 100 pg (SUVmax: 4.37 ± 0.22; %ID / g: 23.89 ± 2.06) either slightly reduced or had no significant effect on tumor uptake. These findings highlight the importance of carefully optimizing non-radioactive antibody dose, as lower doses potentially suppress background while higher doses may interfere with tumor uptake. Co-injection also lead to increased radioactivity in the blood and slightly elevated uptake in kidneys and muscle. Although the exact mechanism is unclear, similar effects have been observed with other radiolabeled antibodies, where co-injection of non-radioactive antibodies prolongs blood circulation, reduces off-target uptake, and improves tumor uptake.43 45This co-injection (carrier-added) or pre-blocking strategy has been reported in several studies to improve tracer biodistribution by saturating antigenexpressing normal tissues.46 47Furthermore, this strategy could be extended to applied to paired therapeutic applications using67Cu-labeled TSHR-Ab, where increased tumor retention and reduced off-target radiation exposure could improve both the efficacy and safety of TSHR-targeted radioligand therapy.

[0179] For advanced and metastatic thyroid cancer, conventional131l and99mTc SPECT scans often fail to detect primary and metastatic lesions. In these cases, [18F]FDG PET imaging is typically recommended.48However, since [18F]FDG is not a target-specific tracer, it often results in false negatives or false positives. In a study, the high uptake of [64Cu]Cu-NOTA-TSHR in TSHR-positive tumors demonstrates its potential as a highly specific radiotracer for imaging advanced metastatic thyroid cancer (FIG. 16B). In contrast, [18F] FDG, the conventional PET radiotracer, exhibited poor tumor uptake (FIG. 16A). The superior tumor targeting of [64Cu]Cu- NOTA-TSHR-Ab is likely attributed to the high affinity of the TSHR-specific antibody (K1-70), enabling precise tumor localization. The significantly higher T / NT ratio further highlights its enhanced tumor contrast, which is crucial for accurate diagnosis and treatment planning. To ensure an accurate comparison between [64Cu]Cu- NOTA-TSHR and [18F] FDG, mice were not co-injected with non-radioactive TSHR-Ab. Therefore, one notable finding is the elevated uptake of [64Cu]Cu-NOTA-TSHR-Ab in the spleen, bone, and liver. Without wishing to be bound by any particular theory, it is thought that this phenomenon is likely due to the metabolism and clearance pathways of whole antibodies. However, this and previous studies indicate that co-injection of non-radioactive antibody can effectively reduce background uptake, suggesting a potential strategy for optimizing imaging contrast.16 43The low kidney uptake of [64Cu]Cu-NOTA-TSHR-Ab, compared to [18F] FDG, is expected, as intact antibodies are primarily cleared via the reticuloendothelial system rather than renal excretion. This characteristic is advantageous in minimizing nephrotoxicity and reducing interference in renal imaging. Clinically, the superior tumor specificity of [64Cu]Cu-NOTA-TSHR-Ab suggests its potential role in diagnosing and staging RAI-resistant,Docket No. 31134 / 2023-456 / PC progressive thyroid cancer, particularly in cases where [18F]FDG fails to detect tumor and metastatic lesions. It also offers a promising approach to identify thyroid cancer patients with TSHR expression who may benefit from TSHR-targeted therapy.Example 11 : Synthesis and characterization of64Cu-NOTA-TSHR-Fab (Construct A65) and64Cu-NOTA-TSHR- ScFv (Construct A66)

[0180] 64Cu-NOTA-TSHR-Fab and64Cu-NOTA-TSHR-scFv were prepared by complexing64CuCl2 with their conjugates, respectively, shown in FIG. 17 and described below. Briefly,64CuCl2 (20-80 MBq in 10-20 piL of 0.05 M HCI aqueous solution) was mixed with 0.1 M ammonium acetate buffer (pH 5.5, 200 pL) to pH 5.3-5.5 at room temperature. The conjugate NOTA-TSHR-Fab or NOTA-TSHR-ScFv (20-90 pig) in 0.2 M NH4AC buffer solution (pH 6.5) was added to the above64Cu-solution and incubated at 37 °C for 60 minutes for radiolabeling. The resulting64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-scFv mixture was purified using a PD10 gel filtration column with PBS (pH=7.4) or normal saline solution as eluent. The radiochemical identity and purity of the final product64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-scFv were determined by radio-TLC (FIGs. 18A’-18A”) and radio-HPLC (FIGs. 18B’-18B”).

[0181] Approximately 3.7 MBq / 100 piL of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR- scFv was mixed with 100 piL of normal saline at room temperature (RT) or 10% fetal bovine serum (FBS) at 37 °C. Aliquots (1-2 pL) were taken at 0, 1, 2, 4, 24, 48, and 72 h and analyzed using a radio-TLC scanner to determine the radiochemical purity (RCP) as an indicator of the stability. The radiochromatograms were compared with the quality control chromatogram for stability analysis, shown in FIGs. 18C’-18C”.Example 12: In vitro evaluation of64Cu-NOTA-TSHR-Fab (Construct A65) and64Cu-NOTA-TSHR-ScFv (Construct A66)

[0182] To assess the targeting potential of64Cu-NOTA-TSHR-Fab and64Cu-NOTA-TSHR-ScFv for TSHR- positive thyroid tumor cells, a series of in vitro uptake and binding affinity studies was conducted using THJ529TrsHfi+or TH J529T^cells. The uptake of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv was significantly higher in THJ529TreHR+cells compared to THJ529T14'7' cells negative for TSHR expression. This uptake was substantially reduced by the addition of an excess of non-radioactive TSHR-Ab, indicating the high cellular specificity of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv binding to TSHR-positive thyroid tumor cells (FIG. 19A’). Furthermore,64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv cell studies demonstrated a nanomolar binding affinity for THJ529TreHR+cells, with a Kd of 13.02 nM (FIG. 19B’) or 4.57 nM (FIG. 19B”) and a Kj of 4.85 nM (FIG. 19C’) or 1.84 nM (FIG. 19C”), respectively.Example 13: In vivo evaluation of64Cu-NOTA-TSHR-Fab (Construct A65) and64Cu-NOTA-TSHR-ScFv (Construct A66)

[0183] Immuno-PET imaging of64Cu-NOTA-TSHR-Fab and64Cu-NOTA-TSHR-ScFv was examined in a subcutaneous tumor NSG mouse model, whose left flank was implanted with a TH J-529TTSHR+tumor (TSHRDocket No. 31134 / 2023-456 / PC positive). The 1 , 4, 18, and 24 h post-injection (p.i.) imaging time points were chosen for serial PET scans after intravenous64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv injection. For blocking imaging, mice were injected with unlabeled TSHR-Ab 1 day prior radiotracer injection. Data from PET / CT imaging were reconstructed and analyzed using MIM version 7.3.5 software (Cleveland, OH, USA). For each image, regions of interest (ROIs) were drawn over the tumor on PET images to calculate the maximum standardized uptake value (SUVmax). Representative coronal projection images of64Cu-NOTA-TSHR-Fab or64Cu-NOTA-TSHR-ScFv PET- CT after injection of 100 piCi of radiotracer in a subcutaneous mouse model with TSHR positive tumor THJ- 529TTSHR+in the left flank (arrow) from 1 hour to 24 hours are shown in FIGs. 20A-20B.64Cu-NOTA-TSHR-Fab and64Cu-NOTA-TSHR-ScFv PET showed high TH J529TTSHR+tumor uptake, which could be blocked by pretreatment with cold antibody K1-70 (FIGs. 20C-20D), demonstrating their imaging specificity. Background signals from organs such as the blood, liver, muscle were low, and kidney were high over 1-24 hours (FIGs. 20A-20D). Collectively, the whole-body PET / CT imaging of64Cu-NOTA-TSHR-Fab in tumor xenograft mice demonstrate that this radiotracer has the potential for PET imaging TSHR expression with reasonable tumor uptake and contrast.Example 14: Ex vivo evaluation of64Cu-NOTA-TSHR-Fab64Cu-NOTA-TSHR-Fab (Construct A65) and64Cu- NOTA-TSHR-ScFv (Construct A66)

[0184] After PET imaging, ex vivo biodistribution experiments for64Cu-NOTA-TSHR-Fab or64Cu-NOTA- TSHR-ScFv were conducted in mice bearing THJ529TTSHR+tumors at 24 h p.i. (n = 3). Tissues, including blood, heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, femur, muscle, brain, and tumors were harvested and weighed, and their radioactivity was measured by a gamma counter. The data were decay- corrected and expressed as the percentage injected dose per gram of tissue (%ID / g), shown in FIG. 21A-21 B. The biodistribution data of both radiotracers at 24 h post-injection were consistent with the results of PET study. Overall, the quantification results obtained from biodistribution studies and PET scans matched well, confirming that quantitative ROI analysis of noninvasive micro-PET / CT scans accurately reflected the distribution of64Cu- NOTA-TSHR-Fab and64Cu-NOTA-TSHR-ScFv in vivo with TSHR antibody coinjection.Example 15: Synthesis and characterization of67Cu-NOTA-TSHR-Ab (Construct A19)

[0185] 67Cu-NOTA-TSHR-Ab was prepared by complexing67CuCl2 with the conjugate NOTA-TSHR-Ab, shown in FIG. 22A and described below. Briefly, ~60 pg of purified NOTA-TSHR-Ab in 0.2 M NH4AC buffer (pH 6.5) was mixed with -740 MBq of67CuCl2, and the pH was adjusted to 5-6 using 0.1 M NH4AC buffer (pH 5.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% sodium chloride, yielding67Cu- NOTA-TSHR-Ab. The radiochemical identity and purity of the final product67Cu-NOTA-TSHR-Ab were determined by radio-TLC (FIG. 22B).Docket No. 31134 / 2023-456 / PCExample 16: Radioimmunotherapy of67Cu-NOTA-TSHR-Ab (Construct A19)

[0186] To evaluate the potential therapeutic capacity of67Cu-NOTA-TSHR-Ab, TH J529TTSHR+tumor-bearing mice were randomly divided into five groups (n = 5): high dose (0.4 mCi), medium dose (0.2 mCi), low dose (0.1 mCi), TSHR-Ab (25 pig), and saline control. Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded one to three times per week. Therapeutic efficacy of67Cu-NOTA-TSHR-Ab (0.1 , 0.2, and 0.4 mCi, with 25 pig for TSHR-antibody K1-70, n= 5) in comparison to the control groups (normal saline or 25 pig for TSHR-antibody K1-70) were presented in FIG. 23A- 23C. The average tumor growth for the mice groups treated with67Cu-NOTA-TSHR-Ab in comparison to the control groups was shown in FIG. 23A. The average body weights for mice group treated with67Cu-NOTA- TSHR-Ab in comparison to the control groups is shown in FIG. 23B. The survival data for mice treated with various doses of67Cu-NOTA-TSHR-Ab in comparison to the control groups is presented in FIG. 23C.Example 17: Synthesis and characterization of177Lu-DOTA-TSHR-Ab (Construct A17)

[0187] 177Lu-DOTA-TSHR-Ab was prepared by complexing177LuCl3 with the conjugate DOTA-TSHR-Ab, shown in FIG. 24A and described below. Briefly, 60-90 pg of purified DOTA-TSHR-Ab in 0.01 M PBS (pH 7.4) was mixed with 30-100 MBq of177LuCl3 and ascorbic acid, and the pH was adjusted to 5-6 using 0.1 M NH4AC buffer (pH 5.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% aqueous sodium chloride, yielding177Lu-DOTA-TSHR-Ab. The radiochemical identity and purity of the final product were determined by instant radio-TLC (FIG. 24B).Example 18: Radioimmunotherapy of177Lu-DOTA-TSHR-Ab (Construct A17)

[0188] To evaluate the potential therapeutic capacity of177Lu-DOTA-TSHR-Ab, TH J529TTSHR+tumor-bearing mice were randomly divided into five groups (n = 5): high dose (0.3 mCi), medium dose (0.2 mCi), low dose (0.1 mCi), TSHR-Ab (25 pig), and saline control. Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded every three days. Therapeutic efficacy of177Lu-DOTA-TSHR-Ab (0.1 , 0.2, and 0.3 mCi, with 25 pig for TSHR-antibody K1-70, n= 5) in comparison to the control groups (normal saline or 25 pig for TSHR-antibody K1-70) is presented in FIG. 25A-25C. The average tumor growth for the mice groups treated with177Lu-DOTA-TSHR-Ab in comparison to the control groups is shown in FIG. 25A. The average body weights for mice group treated with177Lu-DOTA-TSHR-Ab in comparison to the control groups is shown in FIG. 25B. The survival data for mice treated with various doses of177Lu-DOTA- TSHR-Ab in comparison to the control groups is presented in FIG. 25C.Example 19: Synthesis and characterization of225Ac-DOTA-TSHR-Ab (Construct A27)

[0189] 255Ac-DOTA-TSHR-Ab was prepared by complexing225AcCl3 with the conjugate DOTA-TSHR-Ab, shown in FIG. 26A and described below. Briefly, ~80 pg of purified DOTA-TSHR-Ab in 0.2 M NH4OAC (pH 6.5) was mixed with ~3 MBq of225Ac3+and ascorbic acid, and the pH was adjusted to 5-6 using 0.2 M NH4OAC bufferDocket No. 31134 / 2023-456 / PC(pH 6.5). The mixture was incubated at 37 °C for 1 h in a Thermomixer that was set to 500 rpm. After incubation, the reaction mixture was purified via a PD-10 desalting column and eluted with 0.9% sodium chloride, yielding225Ac-DOTA-TSHR-Ab. The radiochemical identity and purity of the final product were determined by instant radio-TLC (FIG. 26B).Example 20: Radioimmunotherapy of255Ac-DOTA-TSHR-Ab (Construct A27)

[0190] To evaluate the potential therapeutic capacity of255Ac-DOTA-TSHR-Ab, THJ529TTSHR+tumor-bearing mice were randomly divided into four groups (n = 5): high dose (20 kBq), medium dose (10 kBq), low dose (5 kBq), TSHR-Ab (25 pig), and saline control. Treatments started when tumor volume reached approximately 100 mm3. Tumor growth and body weight were monitored and recorded every three days. Therapeutic efficacy of255Ac-DOTA-TSHR-Ab (5, 10, and 20 kBq, with 25 pig for TSHR-antibody K1-70, n= 5) in comparison to the control group normal saline are presented in FIG. 27A-27C. The average tumor growth for the mice groups treated with255Ac-DOTA-TSHR-Ab in comparison to the control group is shown in FIG. 25A. The average body weights for mice group treated with255Ac-DOTA-TSHR-Ab in comparison to the control group is shown in FIG. 25B. The survival data for mice treated with various doses of255Ac-DOTA-TSHR-Ab in comparison to the control group is presented in FIG. 25C.REFERENCES1. Kim, J.et al., Nat Rev Endocrinol, 2020. 16(1): p. 17-29.2. French, J.D., Nat Rev Endocrinol, 2020. 16(11): p. 629-641.3. Viola, D., et al., Endocr Relat Cancer, 2016. 23(4): p. R185-205.4. Kogai, T. et al., Pharmacol Ther, 2012. 135(3): p. 355-70.5. Suzuki, K., et al., Biochimie, 1999. 81(4): p. 329-40.6. Rowe, C.W., et al., Endocr Relat Cancer, 2017. 24(6): p. R191-R202.7. Carayon, P., et al., J Clin Endocrinol Metab, 1980. 51(4): p. 915-20.8. Singh, S.P., et al., Endocrinology, 2004. 145(2): p. 1003-1010.9. Marcus, C., et al., AJR Am J Roentgenol, 2014. 202(6): p. 1316-29.10. Kist, J.W., et al., J Nucl Med, 2016. 57(5): p. 701-7.11. Samnick, S., et al., Clin Nucl Med, 2018. 43(3): p. 162-167.12. Corsetti, F., et al., Cancer Biother Radiopharm, 2004. 19(1): p. 57-63.13. Gershengorn, M.C. et al., J Clin Endocrinol Metab, 2012. 97(12): p. 4287-92.14. Sanders, J., et al., Methods Enzymol, 2010. 485: p. 393-420.15. Galli, F., et al., Thyroid, 2014. 24(8): p. 1297-1308.16. Galli, F., et al., Journal of Clinical Medicine, 2021. 10(9).17. Gimblet, G.R., et al., Surgery, 2024. 175(1): p. 199-206.18. Furmaniak, J., et al., Clin Endocrinol (Oxf), 2022. 96(6): p. 878-887.19. Furmaniak, J., et al., Auto Immun Highlights, 2019. 10(1): p. 11.20. Knowles, S.M. et al., J Clin Oncol, 2012. 30(31): p. 3884-92.21. Evans, M., et al., Clinical Endocrinology, 2010. 73(3): p. 404-412.22. Nunez Miguel, R., et al., J Mol Endocrinol, 2023. 70(1).23. Sharma, S.K., et al., Bioconjug Chem, 2021. 32(7): p. 1177-1191.24. Vosjan, M.J., et al., Nat Protoc, 2010. 5(4): p. 739-43.25. Marlow, L.A., et al., J Clin Endocrinol Metab, 2018. 103(9): p. 3169-3182.26. Landa, I., et al., Clin Cancer Res, 2019. 25(10): p. 3141-3151.27. Demeure, M.J., et al., World J Surg, 1992. 16(4): p. 770-6.28. Meares, C.F., et al., Anal Biochem, 1984. 142(1): p. 68-78.29. Szkudlinski, M.W., et al., Physiol Rev, 2002. 82(2): p. 473-502.Docket No. 31134 / 2023-456 / PC 47. . . . 9.Docket No. 31134 / 2023-456 / PC , 103 (9), 3169-3182.

Claims

Docket No. 31134 / 2023-456 / PCWhat is claimed is:1 . A thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical, comprising a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide.

2. The TSHR-targeting radiopharmaceutical of claim 1 , wherein the TSHR-targeting moiety is a small molecule, a protein, a peptide, an antibody, a single-chain variable fragment (scFv), or an antibody fragment (Fab).

3. The TSHR-targeting radiopharmaceutical of claim 1 or 2, wherein the TSHR-targeting moiety is an antibody or an scFv or a Fab thereof.

4. The TSHR-targeting radiopharmaceutical of claim 3, wherein the antibody is a naturally- occurring antibody.

5. The TSHR-targeting radiopharmaceutical of claim 3 or 4, wherein the antibody is an antagonist or an agonist.

6. The TSHR-targeting radiopharmaceutical of any one of claims 3 to 5, wherein the antibody is a monoclonal antibody, a humanized antibody, or a human antibody.

7. The TSHR-targeting radiopharmaceutical of any one of claims 2 to 6, wherein the antibody is K1-70, Human Anti-TSHR Recombinant Antibody (clone K1-70), K1-18, M22, or an scFv or a Fab thereof.

8. The TSHR-targeting radiopharmaceutical of claim 7, wherein the antibody is K1-70, K1-18, or M22.

9. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 8, wherein the chelator moiety comprises a linker moiety, a prosthetic agent, or both.

10. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 9, wherein the chelator moiety comprises a bifunctional chelator.11 . The TSHR-targeting radiopharmaceutical of any one of claims 1 to 10, wherein the chelator moiety comprises desferrioxamine (DFO), 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane tetraacetic acid (DOTA), 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic amide (DOTAM), or sarcophagine (Sar).Docket No. 31134 / 2023-456 / PC12. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 11 , wherein the chelator moiety comprises desferrioxamine.

13. The TSHR-targeting radiopharmaceutical of claim 12, wherein the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, (DFO-Bn-SCN), p-SCN-Bn-NOTA, maleimido-mono-amide-NOTA, maleimido-mono-amide-DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, p-SCN-Bn-DOTAM, DOTAM-NHS-ester, MECOSAR, BisCOSAR, AmBa-Sar, or DiBaSAr.

14. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 13, wherein the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, p-SCN-Bn-NOTA, NOTA-NHS-ester, DOTA-NHS- ester, p-SCN-Bn-DOTAM, or p-SCN-Bn-DOTA.

15. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 14, wherein the chelator moiety comprises N-Succinimidyl 4-[18F]fluorobenzoate (18F-SFB), N-succinimidyl 3-[131l]iodobenzoate (131l-SI B), or N-succinimidyl 3-[211At]astatobenzoate (211At-SAB).

16. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 15, wherein the radionuclide is a positron-emitter, an a-emitter, a p-emitter, an auger emitter, or a y-emitter.

17. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 16, wherein the radionuclide is a positron-emitter.

18. The TSHR-targeting radiopharmaceutical of claim 17, wherein the radionuclide is89Zr,124l,68Ga,640u,610u,52Mn,55Co,44Sc,86Y,11C, or18F.

19. The TSHR-targeting radiopharmaceutical of claim 17 or 18, wherein the radionuclide is89Zr or640u.

20. The TSHR-targeting radiopharmaceutical of any one of claims 17 to 19, wherein the radionuclide is89Zr.

21. The TSHR-targeting radiopharmaceutical of any one of claims 17 to 19, wherein the radionuclide is64Cu.

22. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 16, wherein the radionuclide is an a-emitter.

23. The TSHR-targeting radiopharmaceutical of claim 22, wherein the radionuclide is225Ac,211At,149Tb, or212Pb.Docket No. 31134 / 2023-456 / PC24. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 16, wherein the radionuclide is a p-emitter.

25. The TSHR-targeting radiopharmaceutical of claim 24, where the radionuclide is131l,177Lu, i6iTb, 9oy, 4?Sc,188Re, or67Cu.

26. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 16, wherein the radionuclide is an auger emitter.

27. The TSHR-targeting radiopharmaceutical of claim 26, where the radionuclide is111ln,125l,123l, or201Ti.

28. The TSHR-targeting radiopharmaceutical of any one of claims 1 to 16, wherein the radionuclide is a y-emitter.

29. The TSHR-targeting radiopharmaceutical of claim 28, where the radionuclide is123l or99mTc.

30. A TSHR-targeting radiopharmaceutical as listed in Table 1.31 . 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 thyroid-stimulating hormone receptor-(TSHR- ) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide.

32. The method of claim 31, wherein the TSHR-targeting moiety is a small molecule, a protein, a peptide, an antibody, a single-chain variable fragment (scFv), or an antibody fragment (Fab).

33. The method of claim 31 or 32, wherein the TSHR-targeting moiety is an antibody or an scFv or a Fab thereof.

34. The method of claim 33, wherein the antibody is a naturally-occurring antibody.

35. The method of claim 33 or 34, wherein the antibody is an antagonist or an agonist.

36. The method of any one of claims 33 to 35, wherein the antibody is wherein the antibody is a monoclonal antibody, a humanized antibody or a human antibody.

37. The method of any one of claims 33 to 36, wherein the antibody is K1-70, Human Anti-TSHR Recombinant Antibody (clone K1-70), K1-18, M22, or an scFv or a Fab thereof.Docket No. 31134 / 2023-456 / PC38. The method of claim 37, wherein the antibody is K1-70, K1-18, or M22.

39. The method of any one of claims 31 to 38, wherein the chelator moiety comprises a linker moiety, a prosthetic agent, or both.

40. The method any one of claims 31 to 39, wherein the chelator moiety comprises a bifunctional chelator.41 . The method of any one of claims 31 to 40, wherein the chelator moiety comprises desferrioxamine (DFO), 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic amide (DOTAM), or sarcophagine (Sar).

42. The method of any one of claims 31 to 41 , wherein the chelator moiety comprises desferrioxamine.

43. The method of claim 42, wherein the chelator moiety comprises desferrioxamine-p-benzyl- isothiocyanate, (DFO-Bn-SCN), p-SCN-Bn-NOTA, maleimido-mono-amide-NOTA, maleimido-mono-amide- DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, p-SCN-Bn-DOTAM, DOTAM-NHS-ester, MECOSAR, BisCOSAR, AmBa-Sar, or DiBaSAr.

44. The method of any one of claims 31 to 43, wherein the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, p-SCN-Bn-NOTA, NOTA-NHS-ester, DOTA-NHS-ester, p-SCN-Bn- DOTAM, or p-SCN-Bn-DOTA.

45. The method of any one of claims 30 to 43, wherein the chelator moiety comprises N- Succinimidyl 4-[18F]fluorobenzoate (18F-SFB), N-succinimidyl 3-[131l]iodobenzoate (131I-SI B), or N-succinimidyl 3- [211At]astatobenzoate (211At-SAB).

46. The method of any one of claims 31 to 45, wherein the radionuclide is an a-emitter or a p- emitter.

47. The method of any one of claims 31 to 46, wherein the radionuclide is an a-emitter.

48. The method of claim 47, wherein the radionuclide is225Ac,211At, or212Pb.

49. The method of any one of claims 31 to 46, wherein the radionuclide is a p-emitter.

50. The method of claim 49, where the radionuclide is131l,177Lu,161Tb,90Y,47Sc,188Re, or67Cu.Docket No. 31134 / 2023-456 / PC51 . The method of any one of claims 31 to 50, wherein the TSHR-targeting radiopharmaceutical is the TSHR-targeting radiopharmaceutical of any one of claims 1 to 30.

52. The method of any one of claims 31 to 51 , wherein the disease or disorder is cancer or a thyroid disease.

53. The method of claim 52, wherein the cancer is thyroid cancer.

54. The method of claim 52 or 53, wherein the cancer is differentiated thyroid cancer, papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), insular thyroid carcinoma (ITC), anaplastic thyroid carcinoma (ATC), medullary thyroid carcinoma (MTC), or oncocytic thyroid carcinoma (OTC).

55. The method of claim 52, wherein the thyroid disease is Graves disease or hyperthyroidism.

56. The method of any one of claims 31 to 55, wherein the patient has previously been administered anti-TSHR targeted therapy.

57. The method of claim 56, wherein the anti-TSHR targeted therapy is TSH suppression therapy, TSHR-targeted immunotherapy, TSHR-targeted molecular therapy, TSHR-targeted radiopharmaceutical therapy, or TSHR-targeted chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

58. The method of claim 57, wherein the anti-TSHR targeted therapy is TSHR-targeted CAR T-cell therapy.

59. A method of diagnosing a disease or disorder in a patient, comprising the steps of:(a) administering to the patient a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety; a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide;(b) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours 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 2:1 or greater.

60. A method of treating a disease or disorder in a patient, comprising the steps of:(a) diagnosing a disease or disorder in a patient, comprising I) administering to the patient a thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical comprising: a TSHR-targeting moiety;Docket No. 31134 / 2023-456 / PC a chelator moiety covalently linked to the TSHR-targeting moiety; and a radionuclide;(II) measuring the level of radiation in a first tissue and a second tissue, measured in the patient 0.5 to 168 hours after said administering, said measuring comprising Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET); and(iii) diagnosing the disease or disorder if the ratio of radiation measured in the first tissue compared to the second tissue is 2:1 or greater; and b) treating the disease or disorder by administering to the patient a therapeutically-effective amount of the thyroid-stimulating hormone receptor-(TSHR-) targeting radiopharmaceutical.61 . The method of claim 59 or 60, wherein the TSHR-targeting moiety is a small molecule, protein, a peptide, an antibody, a single-chain variable fragment (scFv), or an antibody fragment (Fab).

62. The method of any one of claims 59 to 61 , wherein the TSHR-targeting moiety is an antibody or an scFv or a Fab thereof.

63. The method of claim 62, wherein the antibody is a naturally-occurring antibody.

64. The method of claim 62 or 63, wherein the antibody is an antagonist or an agonist.

65. The method of any one of claims 61 to 64, wherein the antibody is a monoclonal antibody, a humanized antibody or a human antibody.

66. The method of any one of claims 61 to 65, wherein the antibody is K1-70, Human Anti-TSHR Recombinant Antibody (clone K1-70), K1-18, M22, or an scFv or a Fab thereof.

67. The method of claim 66, wherein the antibody is K1-70, K1-18, or M22.

68. The method of any one of claims 59 to 67, wherein the chelator moiety comprises a linker moiety, a prosthetic agent, or both.

69. The method any one of claims 59 to 68, wherein the chelator moiety comprises a bifunctional chelator.

70. The method of any one of claims 59 to 69, wherein the chelator moiety comprises desferrioxamine (DFO), 1 ,4,7-Triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic amide (DOTAM), or sarcophagine (Sar).71 . The method of any one of claims 59 to 70, wherein the chelator moiety comprises desferrioxamine.Docket No. 31134 / 2023-456 / PC72. The method of claim 71 , wherein the chelator moiety comprises desferrioxamine-p-benzyl- isothiocyanate, (DFO-Bn-SCN), p-SCN-Bn-NOTA, maleimido-mono-amide-NOTA, maleimido-mono-amide- DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, p-SCN-Bn-DOTAM, DOTAM-NHS-ester, MECOSAR, BisCOSAR, AmBa-Sar, or DiBaSAr.

73. The method of any one of claims 59 to 72, wherein the chelator moiety comprises desferrioxamine-p-benzyl-isothiocyanate, p-SCN-Bn-NOTA, NOTA-NHS-ester, DOTA-NHS-ester, p-SCN-Bn- DOTAM, or p-SCN-Bn-DOTA.

74. The method of any one of claims 59 to 73, wherein the chelator moiety comprises N- Succinimidyl 4-[18F]fluorobenzoate (18F-SFB), N-succinimidyl 3-[131l]iodobenzoate (131I-SI B), or N-succinimidyl 3- [211At]astatobenzoate (211At-SAB).

75. The method of any one of claims 59 to 74, wherein the radionuclide is a positron-emitter or a y- emitter.

76. The method of any one of claims 59 to 75, wherein the radionuclide is a positron-emitter.

77. The method of claim 76, wherein the radionuclide is89Zr,124l,68Ga,640u,610u,52Mn,55Co,44Sc,86Y, or18F.

78. The method of claim 76 or 77, wherein the radionuclide is89Zr or640u.

79. The method of any one of claims 76 to 78, wherein the radionuclide is89Zr.

80. The method of any one of claims 76 to 78, wherein the radionuclide is64Cu.81 . The method of any one of claims 59 to 80, wherein the radionuclide is a y-emitter.

82. The method of claim 81 , where the radionuclide is123l or99mTc.

83. The method of any one of claims 59 to 82, wherein the TSHR-targeting radiopharmaceutical is the TSHR-targeting radiopharmaceutical of any one of claims 1 to 30.

84. The method of any one of claims 59 to 83, wherein the disease or disorder is cancer or a thyroid disease.

85. The method of claim 84, wherein the cancer is thyroid cancer.

86. The method of claim 84 or 85, wherein the cancer is differentiated thyroid cancer, papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), insular thyroid carcinoma (ITC), anaplastic thyroid carcinoma (ATC), medullary thyroid carcinoma (MTC), or oncocytic thyroid carcinoma (OTC).Docket No. 31134 / 2023-456 / PC87. The method of claim 84, wherein the thyroid disease is Graves disease or hyperthyroidism.

88. The method of any one of claims 59 to 87, wherein the patient has previously been administered anti-TSHR targeted therapy.

89. The method of claim 88, wherein the anti-TSHR targeted therapy is TSH suppression therapy, TSHR-targeted immunotherapy, TSHR-targeted molecular therapy, TSHR-targeted radiopharmaceutical therapy, or TSHR-targeted chimeric antigen receptor T-cell therapy (CAR T-cell therapy).

90. The method of claim 89, wherein the anti-TSHR targeted therapy is TSHR-targeted CAR T-cell therapy.91 . The method of any one of claims 59 to 90, wherein the first tissue is tumor tissue.

92. The method of any one of claims 59 to 91 , wherein the second tissue is liver tissue, muscle tissue, or blood.

93. The method of any one of claims 59 to 92, wherein the measuring comprises Single-Photon Emission Computed Tomography (SPECT).

94. The method of any one of claims 59 to 93, wherein the measuring comprises Positron Emission Tomography (PET).

95. The method of any one of claims 59 to 94, wherein the TSHR-targeting radiopharmaceutical is co-administered with an unlabeled TSHR-targeting antibody.

96. The method of claim 95, wherein the unlabeled TSHR-targeting antibody is K1-70.

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