Preclinical Evaluation of a New Series of Albumin-Binding 177Lu-Labeled PSMA-Based Low-Molecular-Weight Radiotherapeutics.

Prostate-specific membrane antigen (PSMA)-based low-molecular-weight agents using beta(ß)-particle-emitting radiopharmaceuticals is a new treatment paradigm for patients with metastatic castration-resistant prostate cancer. Although results have been encouraging, there is a need to improve the tumor residence time of current PSMA-based radiotherapeutics. Albumin-binding moieties have been used strategically to enhance the tumor uptake and retention of existing PSMA-based investigational agents. Previously, we developed a series of PSMA-based, ß-particle-emitting, low-molecular-weight compounds. From this series, 177Lu-L1 was selected as the lead agent because of its reduced off-target radiotoxicity in preclinical studies. The ligand L1 contains a PSMA-targeting Lys-Glu urea moiety with an N-bromobenzyl substituent in the ε-amino group of Lys. Here, we structurally modified 177Lu-L1 to improve tumor targeting using two known albumin-binding moieties, 4-(p-iodophenyl) butyric acid moiety (IPBA) and ibuprofen (IBU), and evaluated the effects of linker length and composition. Six structurally related PSMA-targeting ligands (Alb-L1-Alb-L6) were synthesized based on the structure of 177Lu-L1. The ligands were assessed for in vitro binding affinity and were radiolabeled with 177Lu following standard protocols. All 177Lu-labeled analogs were studied in cell uptake and selected cell efficacy studies. In vivo pharmacokinetics were investigated by conducting tissue biodistribution studies for 177Lu-Alb-L2-177Lu-Alb-L6 (2 h, 24 h, 72 h, and 192 h) in male NSG mice bearing human PSMA+ PC3 PIP and PSMA- PC3 flu xenografts. Preliminary therapeutic ratios of the agents were estimated from the area under the curve (AUC0-192h) of the tumors, blood, and kidney uptake values. Compounds were obtained in >98% radiochemical yields and >99% purity. PSMA inhibition constants (Kis) of the ligands were in the ≤10 nM range. The long-linker-based agents, 177Lu-Alb-L4 and 177Lu-Alb-L5, displayed significantly higher tumor uptake and retention (p < 0.001) than the short-linker-bearing 177Lu-Alb-L2 and 177Lu-Alb-L3 and a long polyethylene glycol (PEG) linker-bearing agent, 177Lu-Alb-L6. The area under the curve (AUC0-192h) of the PSMA+ PC3 PIP tumor uptake of 177Lu-Alb-L4 and 177Lu-Alb-L5 were >4-fold higher than 177Lu-Alb-L2, 177Lu-Alb-L3, and 177Lu-Alb-L6, respectively. Also, the PSMA+ PIP tumor uptake (AUC0-192h) of 177Lu-Alb-L2 and 177Lu-Alb-L3 was ~1.5-fold higher than 177Lu-Alb-L6. However, the lowest blood AUC0-192h and kidney AUC0-192h were associated with 177Lu-Alb-L6 from the series. Consequently, 177Lu-Alb-L6 displayed the highest ratios of AUC(tumor)-to-AUC(blood) and AUC(tumor)-to-AUC(kidney) values from the series. Among the other agents, 177Lu-Alb-L4 demonstrated a nearly similar ratio of AUC(tumor)-to-AUC(blood) as 177Lu-Alb-L6. The tumor-to-blood ratio was the dose-limiting therapeutic ratio for all of the compounds. Conclusions: 177Lu-Alb-L4 and 177Lu-Alb-L6 showed high tumor uptake in PSMA+ tumors and tumor-to-blood ratios. The data suggest that linker length and composition can be modulated to generate an optimized therapeutic agent.

Preclinical Development in Radiopharmaceutical Therapy for Prostate Cancer.

Prostate cancer is a leading cause of cancer death in men worldwide. Among the various treatment options, radiopharmaceutical therapy has shown notable success in metastatic, castration-resistant disease. Radiopharmaceutical therapy is a systemic approach that delivers cytotoxic radiation doses precisely to the malignant tumors and/or tumor microenvironment. Therapeutic radiopharmaceuticals are composed of a therapeutic radionuclide and a high-affinity, tumor-targeting carrier molecule. Therapeutic radionuclides used in preclinical prostate cancer studies are primarily α-, ß--, or Auger-electron-emitting radiometals or radiohalogens. Monoclonal antibodies, antibody-derived fragments, peptides, and small molecules are frequently used as tumor-targeting molecules. Over the years, several important membrane-associated proteases and receptors have been identified, validated, and subsequently used for preclinical radiotherapeutic development for prostate cancer. Prostate-specific membrane antigen (PSMA) is the most well-studied prostate cancer-associated protease in preclinical literature. PSMA-targeting radiotherapeutic agents are being investigated using high-affinity antibody- and small-molecule-based agents for safety and efficacy. Early generations of such agents were developed simply by replacing radionuclides of the imaging agents with therapeutic ones. Later, extensive structure-activity relationship studies were conducted to address the safety and efficacy issues obtained from initial patient data. Recent regulatory approval of the 177Lu-labeled low-molecular-weight agent, 177Lu-PSMA-617, is a significant accomplishment. Current preclinical experiments are focused on the structural modification of 177Lu-PSMA-617 and relevant investigational agents to increase tumor targeting and reduce off-target binding and toxicity in healthy organs. While lutetium-177 (177Lu) remains the most widely used radionuclide, radiolabeled analogs with iodine-131 (128I), yttrium-90 (89Y), copper-67 (67Cu), and terbium-161 (161Tb) have been evaluated as potential alternatives in recent years. In addition, agents carrying the α-particle-emitting radiohalogen, astatine-211 (211At), or radiometals, actinium-225 (225Ac), lead-212 (212Pb), radium-223 (223Ra), and thorium-227 (227Th), have been increasingly investigated in preclinical research. Besides PSMA-based radiotherapeutics, other prominent prostate cancer-related proteases, for example, human kallikrein peptidases (HK2 and HK3), have been explored using monoclonal-antibody-(mAb)-based targeting platforms. Several promising mAbs targeting receptors overexpressed on the different stages of prostate cancer have also been developed for radiopharmaceutical therapy, for example, Delta-like ligand 3 (DLL-3), CD46, and CUB domain-containing protein 1 (CDCP1). Progress is also being made using peptide-based targeting platforms for the gastrin-releasing peptide receptor (GRPR), a well-established membrane-associated receptor expressed in localized and metastatic prostate cancers. Furthermore, mechanism-driven combination therapies appear to be a burgeoning area in the context of preclinical prostate cancer radiotherapeutics. Here, we review the current developments related to the preclinical radiopharmaceutical therapy of prostate cancer. These are summarized in two major topics: (1) therapeutic radionuclides and (2) tumor-targeting approaches using monoclonal antibodies, small molecules, and peptides.

Hetero-bivalent agents targeting FAP and PSMA.

PURPOSE: We developed a theranostic radiopharmaceutical that engages two key cell surface proteases, fibroblast activation protein alpha (FAP) and prostate-specific membrane antigen (PSMA), each frequently overexpressed within the tumor microenvironment (TME). The latter is also expressed in most prostate tumor epithelium. To engage a broader spectrum of cancers for imaging and therapy, we conjugated small-molecule FAP and PSMA-targeting moieties using an optimized linker to provide 64Cu-labeled compounds. METHODS: We synthesized FP-L1 and FP-L2 using two linker constructs attaching the FAP and PSMA-binding pharmacophores. We determined in vitro inhibition constants (Ki) for FAP and PSMA. Cell uptake assays and flow cytometry were conducted in human glioma (U87), melanoma (SK-MEL-24), prostate cancer (PSMA + PC3 PIP and PSMA - PC3 flu), and clear cell renal cell carcinoma lines (PSMA + /PSMA - 786-O). Quantitative positron emission tomography/computed tomography (PET/CT) and tissue biodistribution studies were performed using U87, SK-MEL-24, PSMA + PC3 PIP, and PSMA + 786-O experimental xenograft models and the KPC genetically engineered mouse model of pancreatic cancer. RESULTS: 64Cu-FP-L1 and 64Cu-FP-L2 were produced in high radiochemical yields (> 98%) and molar activities (> 19 MBq/nmol). Ki values were in the nanomolar range for both FAP and PSMA. PET imaging and biodistribution studies revealed high and specific targeting of 64Cu-FP-L1 and 64Cu-FP-L2 for FAP and PSMA. 64Cu-FP-L1 displayed more favorable pharmacokinetics than 64Cu-FP-L2. In the U87 tumor model at 2 h post-injection, tumor uptake of 64Cu-FP-L1 (10.83 ± 1.02%ID/g) was comparable to 64Cu-FAPI-04 (9.53 ± 2.55%ID/g). 64Cu-FP-L1 demonstrated high retention 5.34 ± 0.29%ID/g at 48 h in U87 tumor. Additionally, 64Cu-FP-L1 showed high retention in PSMA + PC3 PIP tumor (12.06 ± 0.78%ID/g at 2 h and 10.51 ± 1.82%ID/g at 24 h). CONCLUSIONS: 64Cu-FP-L1 demonstrated high and specific tumor targeting of FAP and PSMA. This compound should enable imaging of lesions expressing FAP, PSMA, or both on the tumor cell surface or within the TME. FP-L1 can readily be converted into a theranostic for the management of heterogeneous tumors.

A Series of PSMA-Targeted Near-Infrared Fluorescent Imaging Agents.

We have synthesized a series of 10 new, PSMA-targeted, near-infrared imaging agents intended for use in vivo for fluorescence-guided surgery (FGS). Compounds were synthesized from the commercially available amine-reactive active NHS ester of DyLight800. We altered the linker between the PSMA-targeting urea moiety and the fluorophore with a view to improve the pharmacokinetics. Chemical yields for the conjugates ranged from 51% to 86%. The Ki values ranged from 0.10 to 2.19 nM. Inclusion of an N-bromobenzyl substituent at the ε-amino group of lysine enhanced PSMA+ PIP tumor uptake, as did hydrophilic substituents within the linker. The presence of a polyethylene glycol chain within the linker markedly decreased renal uptake. In particular, DyLight800-10 demonstrated high specific uptake relative to background signal within kidney, confirmed by immunohistochemistry. These compounds may be useful for FGS in prostate, renal or other PSMA-expressing cancers.

An Improved 211At-Labeled Agent for PSMA-Targeted α-Therapy.

α-Particle emitters targeting the prostate-specific membrane antigen (PSMA) proved effective in treating patients with prostate cancer who were unresponsive to the corresponding ß-particle therapy. 211At is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new 211At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. Methods: A small series of 125I-labeled compounds was synthesized from tin precursors to evaluate the effect of the location of the radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, 211At-3-Lu was selected and evaluated in cell uptake and internalization studies, and biodistribution and PSMA-expressing (PSMA+) PC3 PIP tumor growth control were evaluated in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-mo) toxicity study was performed for 211At-3-Lu, including tissue chemistries and histopathology. Results: The radiochemical yield of 211At-3-Lu was 17.8% ± 8.2%. Lead compound 211At-3-Lu demonstrated total uptake within PSMA+ PC3 PIP cells of 13.4 ± 0.5% of the input dose after 4 h of incubation, with little uptake in control cells. In SCID mice, 211At-3-Lu provided uptake that was 30.6 ± 4.8 percentage injected dose per gram (%ID/g) in PSMA+ PC3 PIP tumor at 1 h after injection, and this uptake decreased to 9.46 ± 0.96 %ID/g by 24 h. Tumor-to-salivary gland and tumor-to-kidney ratios were 129 ± 99 at 4 h and 130 ± 113 at 24 h, respectively. Deastatination was not significant (stomach, 0.34 ± 0.20 %ID/g at 4 h). Dose-dependent survival was demonstrated at higher doses (>1.48 MBq) in both flank and metastatic models. There was little off-target toxicity, as demonstrated by hematopoietic stability, unchanged tissue chemistries, weight gain rather than loss throughout treatment, and favorable histopathologic findings. Conclusion: Compound 211At-3-Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer.

Preclinical Evaluation of 213Bi- and 225Ac-Labeled Low-Molecular-Weight Compounds for Radiopharmaceutical Therapy of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical therapy is a new option for patients with advanced prostate cancer refractory to other treatments. Previously, we synthesized a ß-particle-emitting low-molecular-weight compound, 177Lu-L1 which demonstrated reduced off-target effects in a xenograft model of prostate cancer. Here, we leveraged that scaffold to synthesize α-particle-emitting analogs of L1, 213Bi-L1 and 225Ac-L1, to evaluate their safety and cell kill effect in PSMA-positive (+) xenograft models. Methods: The radiochemical synthesis, cell uptake, cell kill, and biodistribution of 213Bi-L1 and 225Ac-L1 were evaluated. The efficacy of 225Ac-L1 was determined in human PSMA+ subcutaneous and micrometastatic models. Subacute toxicity at 8 wk and chronic toxicity at 1 y after administration were evaluated for 225Ac-L1. The absorbed radiation dose of 225Ac-L1 was determined using the biodistribution data and α-camera imaging. Results:213Bi- and 225Ac-L1 demonstrated specific cell uptake and cell kill in PSMA+ cells. The biodistribution of 213Bi-L1 and 225Ac-L1 revealed specific uptake of radioactivity within PSMA+ lesions. Treatment studies of 225Ac-L1 demonstrated activity-dependent, specific inhibition of tumor growth in the PSMA+ flank tumor model. 225Ac-L1 also showed an increased survival benefit in the micrometastatic model compared with 177Lu-L1. Activity-escalated acute and chronic toxicity studies of 225Ac-L1 revealed off-target radiotoxicity, mainly in kidneys and liver. The estimated maximum tolerated activity was about 1 MBq/kg. α-Camera imaging of 225Ac-L1 revealed high renal cortical accumulation at 2 h followed by fast clearance at 24 h. Conclusion:225Ac-L1 demonstrated activity-dependent efficacy with minimal treatment-related organ radiotoxicity. 225Ac-L1 is a promising therapeutic for further clinical evaluation.

Preclinical Evaluation of 203/212Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203Pb-L1-203Pb-L5 were performed on mice bearing PSMA(+) PC3 PIP and PSMA(-) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212Pb-L2 was evaluated in PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor models and in the PSMA(+) luciferase-expressing micrometastatic model. 212Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203Pb-L1-203Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203Pb-L1-203Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203Pb-L5 (31.0, 15.2) and lowest for 203Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203Pb-L3 (3.2) and 203Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(+) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(+) flank tumor model. 212Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion:203Pb-L1-203Pb-L5 demonstrated favorable pharmacokinetics for 212Pb-based TRT. The antitumor efficacy of 212Pb-L2 supports the corresponding 203Pb/212Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

177Lu-labeled low-molecular-weight agents for PSMA-targeted radiopharmaceutical therapy.

PURPOSE: To develop a prostate-specific membrane antigen (PSMA)-targeted radiotherapeutic for metastatic castration-resistant prostate cancer (mCRPC) with optimized efficacy and minimized toxicity employing the ß-particle radiation of 177Lu. METHODS: We synthesized 14 new PSMA-targeted, 177Lu-labeled radioligands (177Lu-L1-177Lu-L14) using different chelating agents and linkers. We evaluated them in vitro using human prostate cancer PSMA(+) PC3 PIP and PSMA(-) PC3 flu cells and in corresponding flank tumor models. Efficacy and toxicity after 8 weeks were evaluated at a single administration of 111 MBq for 177Lu-L1, 177Lu-L3, 177Lu-L5 and 177Lu-PSMA-617. Efficacy of 177Lu-L1 was further investigated using different doses, and long-term toxicity was determined in healthy immunocompetent mice. RESULTS: Radioligands were produced in high radiochemical yield and purity. Cell uptake and internalization indicated specific uptake only in PSMA(+) PC3 cells. 177Lu-L1, 177Lu-L3 and 177Lu-L5 demonstrated comparable uptake to 177Lu-PSMA-617 and 177Lu-PSMA-I&T in PSMA-expressing tumors up to 72 h post-injection. 177Lu-L1, 177Lu-L3 and 177Lu-L5 also demonstrated efficient tumor regression at 8 weeks. 177Lu-L1 enabled the highest survival rate. Necropsy studies of the treated group at 8 weeks revealed subacute damage to lacrimal glands and testes. No radiation nephropathy was observed 1 year post-treatment in healthy mice receiving 111 MBq of 177Lu-L1, most likely related to the fast renal clearance of this agent. CONCLUSIONS: 177Lu-L1 is a viable clinical candidate for radionuclide therapy of PSMA-expressing malignancies because of its high tumor-targeting ability and low off-target radiotoxic effects.

Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a Murine Model of Prostate Cancer.

The prostate-specific membrane antigen (PSMA) is a validated target for detection and management of prostate cancer (PC). It has also been utilized for targeted drug delivery through antibody-drug conjugates and polymeric micelles. Polyamidoamine (PAMAM) dendrimers are emerging as a versatile platform in a number of biomedical applications due to their unique physicochemical properties, including small size, large number of reactive terminal groups, bulky interior void volume, and biocompatibility. Here, we report the synthesis of generation 4 PSMA-targeted PAMAM dendrimers [G4(MP-KEU)] and evaluation of their targeting properties in vitro and in vivo using an experimental model of PC. A facile, one-pot synthesis gave nearly neutral nanoparticles with a narrow size distribution of 5 nm in diameter and a molecular weight of 27.3 kDa. They exhibited in vitro target specificity with a dissociation constant ( Kd) of 0.32 ± 0.23 µm and preferential accumulation in PSMA+ PC3 PIP tumors versus isogenic PSMA- PC3 flu tumors. Positron emission tomography-computed tomography imaging and ex vivo biodistribution studies of dendrimers radiolabeled with 64Cu, [64Cu]G4(MP-KEU), demonstrated high accumulation in PSMA+ PC3 PIP tumors at 24 h post-injection (45.83 ± 20.09% injected dose per gram of tissue, %ID/g), demonstrating a PSMA+ PC3 PIP/PSMA- PC3 flu ratio of 7.65 ± 3.35. Specific accumulation of G4(MP-KEU) and [64Cu]G4(MP-KEU) in PSMA+ PC3 PIP tumors was inhibited by the known small-molecule PSMA inhibitor, ZJ-43. On the contrary, G4(Ctrl), control dendrimers without PSMA-targeting moieties, showed comparable low accumulation of ∼1%ID/g in tumors irrespective of PSMA expression, further confirming PSMA+ tumor-specific uptake of G4(MP-KEU). These results suggest that G4(MP-KEU) may represent a suitable scaffold by which to target PSMA-expressing tissues with imaging and therapeutic agents.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/µg (60.54 ± 20 µCi/µg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Salicylic Acid-Based Polymeric Contrast Agents for Molecular Magnetic Resonance Imaging of Prostate Cancer.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is an innovative molecular imaging technique in which contrast agents are labeled by saturating their exchangeable proton spins by radio-frequency irradiation. Salicylic acid and its analogues are a promising class of highly sensitive, diamagnetic CEST agents. Herein, polymeric agents grafted with salicylic acid moieties and a known high-affinity ligand targeting prostate-specific membrane antigen in approximately 10:1 molar ratio were synthesized to provide sufficient MRI sensitivity and receptor specificity. The proton-exchange properties of the contrast agent in solution and in an experimental murine model are reported to demonstrate the feasibility of receptor-targeted CEST MRI of prostate cancer. Furthermore, the CEST imaging data were validated with an 111 In-labeled analogue of the agent by in vivo single photon emission computed tomographic imaging and tissue biodistribution studies.

A dextran-based probe for the targeted magnetic resonance imaging of tumours expressing prostate-specific membrane antigen.

Safe imaging agents able to render the expression and distribution of cancer receptors, enzymes or other biomarkers would facilitate clinical screening of the disease. Here, we show that diamagnetic dextran particles coordinated to a urea-based targeting ligand for prostate-specific membrane antigen (PSMA) enable targeted magnetic resonance imaging (MRI) of the PSMA receptor. In a xenograft model of prostate cancer, micromolar concentrations of the dextran -ligand probe provided sufficient signal to specifically detect PSMA-expressing tumours via chemical exchange saturation transfer MRI. The dextran-based probe could be detected via the contrast originating from dextran hydroxyl protons, thereby avoiding the need of chemical substitution for radioactive or metallic labelling. Because dextrans are currently used clinically, dextran-based contrast agents may help extend receptor-targeted imaging to clinical MRI.

[64Cu]XYIMSR-06: A dual-motif CAIX ligand for PET imaging of clear cell renal cell carcinoma.

Carbonic anhydrase IX (CAIX) is a cell surface enzyme that is over-expressed in approximately 95% of cases of clear cell renal cell carcinoma (ccRCC), the most common renal cancer. We synthesized and performed in vitro and in vivo evaluation of a dual-motif ligand, [64Cu]XYIMSR-06, for imaging CAIX expression on ccRCC tumors using positron emission tomography (PET). [64Cu]XYIMSR-06 was generated in yields of 51.0 ± 4.5% (n=5) and specific activities of 4.1 - 8.9 GBq/µmol (110-240 Ci/mmol). Tumor was visualized on PET images by 1 h post-injection with high tumor-to-background levels (>100 tumor-to-blood and -muscle) achieved within 24 h. Biodistribution studies demonstrated a maximum tumor uptake of 19.3% injected dose per gram of radioactivity at 4 h. Tumor-to-blood, -muscle and -kidney ratios were 129.6 ± 18.8, 84.3 ± 21.0 and 2.1 ± 0.3, respectively, at 8 h post-injection. At 24 h a tumor-to-kidney ratio of 7.1 ± 2.5 was achieved. These results indicate pharmacokinetics superior to those of previously reported imaging agents binding to CAIX. [64Cu]XYIMSR-06 is a new low-molecular-weight PET ligand targeting CAIX, which can image localized and metastatic ccRCC.

Imaging, Biodistribution, and Dosimetry of Radionuclide-Labeled PD-L1 Antibody in an Immunocompetent Mouse Model of Breast Cancer.

The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radionuclide Indium-111 ((111)In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of (111)In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Coinjection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of (111)In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of (111)In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy.

Nanoconjugation of PSMA-Targeting Ligands Enhances Perinuclear Localization and Improves Efficacy of Delivered Alpha-Particle Emitters against Tumor Endothelial Analogues.

This study aims to evaluate the effect on killing efficacy of the intracellular trafficking patterns of α-particle emitters by using different radionuclide carriers in the setting of targeted antivascular α-radiotherapy. Nanocarriers (lipid vesicles) targeted to the prostate-specific membrane antigen (PSMA), which is unique to human neovasculature for a variety of solid tumors, were loaded with the α-particle generator actinium-225 and were compared with a PSMA-targeted radiolabeled antibody. Actinium-225 emits a total of four α-particles per decay, providing highly lethal and localized irradiation of targeted cells with minimal exposure to surrounding healthy tissues. Lipid vesicles were derivatized with two types of PSMA-targeting ligands: a fully human PSMA antibody (mAb) and a urea-based, low-molecular-weight agent. Target selectivity and extent of internalization were evaluated on monolayers of human endothelial cells (HUVEC) induced to express PSMA in static incubation conditions and in a flow field. Both types of radiolabeled PSMA-targeted vesicles exhibit similar killing efficacy, which is greater than the efficacy of the radiolabeled control mAb when compared on the basis of delivered radioactivity per cell. Fluorescence confocal microscopy demonstrates that targeted vesicles localize closer to the nucleus, unlike antibodies which localize near the plasma membrane. In addition, targeted vesicles cause larger numbers of dsDNAs per nucleus of treated cells compared with the radiolabeled mAb. These findings demonstrate that radionuclide carriers, such as PSMA-targeted lipid-nanocarriers, which localize close to the nucleus, increase the probability of α-particle trajectories crossing the nuclei, and, therefore, enhance the killing efficacy of α-particle emitters.

Ultrahigh affinity Raman probe for targeted live cell imaging of prostate cancer.

Precise visualization of tumor margins with characterization of microscopic tumor invasion are unmet needs in prostate oncology that demand approaches with high sensitivity and specificity. To address those needs we report surface-enhanced Raman scattering (SERS) based optical imaging for prostate cancer using a combination of live cell Raman microscopy, optimally engineered SERS tags and a urea-based small-molecule inhibitor of prostate-specific membrane antigen (PSMA) as a targeting moiety. We develop gold nanostar based SERS agents that offer ultrahigh binding affinity to PSMA with nearly four orders of magnitude lower IC50 values in relation to existing clinical imaging agents. This combination enables selective recognition of prostate cancer cells, and facilitates quantitative and photostable Raman measurements. Using Raman microscopy to analyze phenotypically similar prostate cancer cell lines differing only in PSMA expression, we demonstrate facile, site-selective recognition using as low as 20 pM of the SERS agent for imaging, opening the door for spectroscopic detection of prostate and other PSMA-expressing tumors in vivo.

Imaging of carbonic anhydrase IX with an 111In-labeled dual-motif inhibitor.

We developed a new scaffold for radionuclide-based imaging and therapy of clear cell renal cell carcinoma (ccRCC) targeting carbonic anhydrase IX (CAIX). Compound XYIMSR-01, a DOTA-conjugated, bivalent, low-molecular-weight ligand, has two moieties that target two separate sites on CAIX, imparting high affinity. We synthesized [111In]XYIMSR-01 in 73.8-75.8% (n = 3) yield with specific radioactivities ranging from 118 - 1,021 GBq/µmol (3,200-27,600 Ci/mmol). Single photon emission computed tomography of [111In]XYIMSR-01 in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors revealed radiotracer uptake in tumor as early as 1 h post-injection. Biodistribution studies demonstrated 26% injected dose per gram of radioactivity within tumor at 1 h. Tumor-to-blood, muscle and kidney ratios were 178.1 ± 145.4, 68.4 ± 29.0 and 1.7 ± 1.2, respectively, at 24 h post-injection. Retention of radioactivity was exclusively observed in tumors by 48 h, the latest time point evaluated. The dual targeting strategy to engage CAIX enabled specific detection of ccRCC in this xenograft model, with pharmacokinetics surpassing those of previously described radionuclide-based probes against CAIX.

Synthesis and Evaluation of Gd(III) -Based Magnetic Resonance Contrast Agents for Molecular Imaging of Prostate-Specific Membrane Antigen.

Magnetic resonance (MR) imaging is advantageous because it concurrently provides anatomic, functional, and molecular information. MR molecular imaging can combine the high spatial resolution of this established clinical modality with molecular profiling in vivo. However, as a result of the intrinsically low sensitivity of MR imaging, high local concentrations of biological targets are required to generate discernable MR contrast. We hypothesize that the prostate-specific membrane antigen (PSMA), an attractive target for imaging and therapy of prostate cancer, could serve as a suitable biomarker for MR-based molecular imaging. We have synthesized three new high-affinity, low-molecular-weight Gd(III) -based PSMA-targeted contrast agents containing one to three Gd(III)  chelates per molecule. We evaluated the relaxometric properties of these agents in solution, in prostate cancer cells, and in an in vivo experimental model to demonstrate the feasibility of PSMA-based MR molecular imaging.

Design and assembly of supramolecular dual-modality nanoprobes.

We report the design and synthesis of self-assembling dual-modality molecular probes containing both a fluorophore for optical imaging and a metal ion chelator for imaging with MRI or radionuclide methods. These molecular probes can spontaneously associate into spherical nanoparticles under physiological conditions. We demonstrate the use of these supramolecular nanoprobes for live-cell optical imaging, as well as their potential use as MRI contrast agents after complexation with gadolinium. Our results suggest that self-assembly into supramolecular nanoprobes presents an effective means to enhance and tune the relaxivities of molecular probes.