A third generation PSMA-targeted agent [211At]YF2: Synthesis and in vivo evaluation.

INTRODUCTION: Targeted α-particle therapy agents have shown promising responses in patients who have developed resistance to ß--particle emitting radionuclides, albeit off-target toxicity remains a concern. Astatine-211 emits only one α-particle per decay and may alleviate the toxicity from α-emitting daughter radionuclides. Previously, we developed the low-molecular-weight PSMA-targeted agent [211At]L3-Lu that showed suitable therapeutic efficacy and was well tolerated in mice. Although [211At]L3-Lu had good characteristics, we now have evaluated a closely related analogue, [211At]YF2, to determine the better molecule for clinical translation. METHODS: The tin precursors and unlabeled iodo standards for [211At]YF2 and [211At]L3-Lu each were synthesized and a new one-step labeling method was developed to produce [211At]YF2 and [211At]L3-Lu from the respective tin precursor. RCY and RCP were determined using RP-HPLC. Cell uptake, internalization and in vitro cell-killing (MTT) assays were performed on PSMA+ PC-3 PIP cells in parallel experiments to compare [211At]YF2 and [211At]L3-Lu directly. A paired-label biodistribution study was performed in athymic mice with subcutaneous PSMA-positive PC-3 PIP xenografts as a head-to-head comparison of [131I]YF2 and [125I]L3-Lu. The tissue distribution of [211At]YF2 and [211At]L3-Lu were determined individually in the same animal model. RESULTS: The syntheses of tin precursors and unlabeled iodo standards were accomplished in reasonable yields. A streamlined and scalable radiolabeling method (1 h total synthesis time) was developed for the radiosynthesis of both [211At]YF2 and [211At]L3-Lu with 86 ± 7 % (n = 10) and 87 ± 5 % (n = 7) RCY, respectively, and > 95 % RCP for both. The maximum activity of [211At]YF2 produced to date was 666 MBq. An alternative method that did not involve HPLC purification was developed that provided similar RCY and RCP. Significantly higher cell uptake, internalization and cytotoxicity was seen for [211At]YF2 compared with [211At]L3-Lu. Significantly higher uptake and longer retention in tumor was seen for [131I]YF2 than for co-administered [125I]L3-Lu, while considerably higher renal uptake was seen for [131I]YF2. The biodistribution of [211At]YF2 was consistent with that of [131I]YF2. CONCLUSION: [211At]YF2 exhibited higher cellular uptake, internalization and cytotoxicity than [211At]L3-Lu on PSMA-positive PC3 PIP cells. Likewise, higher uptake and longer retention in tumor was seen for [211At]YF2. Experiments to evaluate the dosimetry and therapeutic efficacy of [211At]YF2 are under way.

PSMA-reactive NB7 single domain antibody fragment: A potential scaffold for developing prostate cancer theranostics.

INTRODUCTION: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211At-labeled analogues. METHODS: [*I]SGMIB, iso-[*I]SGMIB, [211At]SAGMB, and iso-[211At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA+ PC3 PIP and PSMA- PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA+ PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His6-tag (NB7H6) and labeled with iso-[125I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131I- vs 211At-labeled NB7 conjugates and c) [125I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [131I]YF2. RESULTS: All NB7 radioconjugates bound specifically to PSMA with dissociation constants, Kd, in the low nM range (1.4-6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[125I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His6-tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [131I]SGMIB-NB7-to-iso-[125I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131I suggesting greater dehalogenation for [211At]SAGMB-NB7H6. Finally, co-administration of [125I]SGMIB-NB7H6 and [131I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate. CONCLUSION: NB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches.

Effective Treatment of Human Breast Carcinoma Xenografts with Single-Dose 211At-Labeled Anti-HER2 Single-Domain Antibody Fragment.

Single-domain antibody fragments (sdAbs) are attractive for targeted α-particle therapy, particularly with 211At, because of their rapid accumulation in tumor and clearance from normal tissues. Here, we evaluate the therapeutic potential of this strategy with 5F7 and VHH_1028-2 sdAbs that bind with high affinity to domain IV of human epidermal growth factor receptor type 2 (HER2). Methods: The HER2-specific sdAbs and HER2-irrelevant VHH_2001 were labeled using N-succinimidyl-3-211At-astato-5-guanidinomethyl benzoate (iso-211At-SAGMB). The cytotoxicity of iso- 211At-SAGMB-5F7 and iso- 211At-SAGMB-VHH_2001 were compared on HER2-expressing BT474 breast carcinoma cells. Three experiments in mice with subcutaneous BT474 xenografts were performed to evaluate the therapeutic effectiveness of single doses of iso- 211At-SAGMB-5F7 (0.7-3.0 MBq), iso- 211At-SAGMB-VHH_1028 (1.0-3.0 MBq), and iso- 211At-SAGMB-VHH_1028 and iso- 211At-SAGMB-VHH_2001 (∼1.0 MBq). Results: Clonogenic survival of BT474 cells was reduced after exposure to iso- 211At-SAGMB-5F7 (D0 = 1.313 kBq/mL) whereas iso- 211At-SAGMB-VHH_2001 was ineffective. Dose-dependent tumor growth inhibition was observed with 211At-labeled HER2-specific 5F7 and VHH_1028 but not with HER2-irrelevant VHH_2001. At the 3.0-MBq dose, complete tumor regression was seen in 3 of 4 mice treated with iso- 211At-SAGMB-5F7 and 8 of 11 mice treated with iso- 211At-SAGMB-VHH_1028; prolongation in median survival was 495% and 414%, respectively. Conclusion: Combining rapidly internalizing, high-affinity HER2-targeted sdAbs with the iso- 211At-SAGMB residualizing prosthetic agent is a promising strategy for targeted α-particle therapy of HER2-expressing cancers.

Site-Specific Radiohalogenation of a HER2-Targeted Single-Domain Antibody Fragment Using a Novel Residualizing Prosthetic Agent.

Because of their rapid tumor accumulation and normal tissue clearance, single-domain antibody fragments (sdAbs) are an attractive vehicle for developing radiotherapeutics labeled with the α-emitter 211At. Herein, we have evaluated iso-[211At]AGMB-PODS, a prosthetic agent that combines a functionality for residualizing radiohalogens with a phenyloxadiazolyl methylsulfone (PODS) moiety for site-specific sdAb conjugation. Iso-[211At]AGMB-PODS and its radioiodinated analogue were evaluated for thiol-selective conjugation to anti-HER2 5F7 sdAb bearing a C-terminus GGC tail. Both radiohalogenated PODS-5F7GGC conjugates were synthesized in good radiochemical yields and retained high binding affinity on HER2-positive BT474 breast carcinoma cells. Iso-[211At]AGMB-PODS-5F7GGC was considerably more stable in vitro than its maleimide analogue in the presence of cysteine and human serum albumin (HSA) and exhibited excellent tumor uptake and high in vivo stability. Superior tumor-to-kidney activity ratios were seen for both radiohalogenated PODS-5F7GGC conjugates compared with [177Lu]Lu-DOTA-PODS-5F7GGC. These results suggest that iso-[211At]AGMB-PODS-5F7GGC warrants further evaluation for the treatment of HER2-expressing malignancies.

Low-level whole-brain radiation enhances theranostic potential of single-domain antibody fragments for human epidermal growth factor receptor type 2 (HER2)-positive brain metastases.

Background: Single-domain antibody fragments (aka VHH, ~ 13 kDa) are promising delivery systems for brain tumor theranostics; however, achieving efficient delivery of VHH to intracranial lesions remains challenging due to the tumor-brain barrier. Here, we evaluate low-dose whole-brain irradiation as a strategy to increase the delivery of an anti- human epidermal growth factor receptor type 2 (HER2) VHH to breast cancer-derived intracranial tumors in mice. Methods: Mice with intracranial HER2-positive BT474BrM3 tumors received 10-Gy fractionated cranial irradiation and were evaluated by noninvasive imaging. Anti-HER2 VHH 5F7 was labeled with 18F, administered intravenously to irradiated mice and controls, and PET/CT imaging was conducted periodically after irradiation. Tumor uptake of 18F-labeled 5F7 in irradiated and control mice was compared by PET/CT image analysis and correlated with tumor volumes. In addition, longitudinal dynamic contrast-enhanced MRI (DCE-MRI) was conducted to visualize and quantify the potential effects of radiation on tumor perfusion and permeability. Results: Increased 18F-labeled 5F7 intracranial tumor uptake was observed with PET in mice receiving cranial irradiation, with maximum tumor accumulation seen approximately 12 days post initial radiation treatment. No radiation-induced changes in HER2 expression were detected by Western blot, flow cytometry, or on tissue sections. DCE-MRI imaging demonstrated transiently increased tumor perfusion and permeability after irradiation, consistent with the higher tumor uptake of 18F-labeled anti-HER2 5F7 in irradiated mice. Conclusion: Low-level brain irradiation induces dynamic changes in tumor vasculature that increase the intracranial tumor delivery of an anti-HER2 VHH, which could facilitate the use of radiolabeled VHH to detect, monitor, and treat HER2-expressing brain metastases.

Preclinical Development of [211At]meta- astatobenzylguanidine ([211At]MABG) as an Alpha Particle Radiopharmaceutical Therapy for Neuroblastoma.

PURPOSE: [131I]meta-iodobenzylguanidine ([131I]MIBG) is a targeted radiotherapeutic administered systemically to deliver beta particle radiation in neuroblastoma. However, relapses in the bone marrow are common. [211At]meta-astatobenzylguanidine ([211At] MABG) is an alpha particle emitter with higher biological effectiveness and short path length which effectively sterilizes microscopic residual disease. Here we investigated the safety and antitumor activity [211At]MABG in preclinical models of neuroblastoma. EXPERIMENTAL DESIGN: We defined the maximum tolerated dose (MTD), biodistribution, and toxicity of [211At]MABG in immunodeficient mice in comparison with [131I]MIBG. We compared the antitumor efficacy of [211At]MABG with [131I]MIBG in three murine xenograft models. Finally, we explored the efficacy of [211At]MABG after tail vein xenografting designed to model disseminated neuroblastoma. RESULTS: The MTD of [211At]MABG was 66.7 MBq/kg (1.8 mCi/kg) in CB17SC scid-/- mice and 51.8 MBq/kg (1.4 mCi/kg) in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Biodistribution of [211At]MABG was similar to [131I]MIBG. Long-term toxicity studies on mice administered with doses up to 41.5 MBq/kg (1.12 mCi/kg) showed the radiotherapeutic to be well tolerated. Both 66.7 MBq/kg (1.8 mCi/kg) single dose and fractionated dosing 16.6 MBq/kg/fraction (0.45 mCi/kg) × 4 over 11 days induced marked tumor regression in two of the three models studied. Survival was significantly prolonged for mice treated with 12.9 MBq/kg/fraction (0.35 mCi/kg) × 4 doses over 11 days [211At]MABG in the disseminated disease (IMR-05NET/GFP/LUC) model (P = 0.003) suggesting eradication of microscopic disease. CONCLUSIONS: [211At]MABG has significant survival advantage in disseminated models of neuroblastoma. An alpha particle emitting radiopharmaceutical may be effective against microscopic disseminated disease, warranting clinical development.

Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers.

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

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.

Gold Nanostars: A Novel Platform for Developing 211At-Labeled Agents for Targeted Alpha-Particle Therapy.

AIM: To develop an innovative 211At nanoplatform with high radiolabeling efficiency and low in vivo deastatination for future targeted alpha-particle therapy (TAT) to treat cancer. METHODS: Star-shaped gold nanoparticles, gold nanostars (GNS), were used as the platform for 211At radiolabeling. Radiolabeling efficiency under different reaction conditions was tested. Uptake in the thyroid and stomach after systemic administration was used to evaluate the in vivo stability of 211At-labeled GNS. A subcutaneous U87MG human glioma xenograft murine model was used to preliminarily evaluate the therapeutic efficacy of 211At-labeled GNS after intratumoral administration. RESULTS: The efficiency of labeling GNS with 211At was almost 100% using a simple and rapid synthesis process that was completed in only 1 min. In vitro stability test in serum showed that more than 99% of the 211At activity remained on the GNS after 24 h incubation at 37°C. In vivo biodistribution results showed low uptake in the thyroid (0.44-0.64%ID) and stomach (0.21-0.49%ID) between 0.5 and 21 h after intravenous injection, thus indicating excellent in vivo stability of 211At-labeled GNS. The preliminary therapeutic efficacy study demonstrated that 211At labeled GNS substantially reduced tumor growth (P < 0.001; two-way ANOVA) after intratumoral administration. CONCLUSION: The new 211At radiolabeling strategy based on GNS has the advantages of a simple process, high labeling efficiency, and minimal in vivo dissociation, making it an attractive potential platform for developing TAT agents that warrants further evaluation in future preclinical studies directed to evaluating prospects for clinical translation.

Labeling single domain antibody fragments with 18F using a novel residualizing prosthetic agent - N-succinimidyl 3-(1-(2-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate.

INTRODUCTION: Labeling single domain antibody fragments (sdAbs) with 18F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations. METHODS: [18F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [18F]RL-III-5F7 also was performed. RESULTS: Radiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [18F]RL-III-5F7 in the intracranial model. CONCLUSIONS: Although higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.

Site-Specific and Residualizing Linker for 18F Labeling with Enhanced Renal Clearance: Application to an Anti-HER2 Single-Domain Antibody Fragment.

Single-domain antibody fragments (sdAbs) are promising vectors for immuno-PET; however, better methods for labeling sdAbs with 18F are needed. Herein, we evaluate a site-specific strategy using an 18F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-18F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (18F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-125I-iodobenzoate (iso-125I-SGMIB). The 2 labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. Small-animal PET/CT imaging after administration of 18F-5F7GGC in the above mouse model was also performed. Results:18F-5F7GGC was synthesized in an overall radiochemical yield of 8.9% ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for 18F-5F7GGC was similar to that for coincubated iso-125I-SGMIB-5F7. Likewise, the uptake of 18F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-125I-SGMIB-5F7; however, 18F-5F7GGC exhibited significantly more rapid clearance from the kidney. Small-animal PET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. Conclusion: This site-specific and residualizing 18F-labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immuno-PET.

Synthesis and preliminary evaluation of 211At-labeled inhibitors of prostate-specific membrane antigen for targeted alpha particle therapy of prostate cancer.

INTRODUCTION: The high potency and short tissue range of α-particles are attractive features for targeted radionuclide therapy, particularly for cancers with micro-metastases. In the current study, we describe the synthesis of a series of 211At-labeled prostate-specific membrane antigen (PSMA) inhibitors and their preliminary evaluation as potential agents for metastatic prostate cancer treatment. METHODS: Four novel Glu-urea based PSMA ligands containing a trialkyl stannyl group were synthesized and labeled with 211At, and for comparative purposes, 131I, via halodestannylation reactions with N-chlorosuccinimide as the oxidant. A PSMA inhibitory assay was performed to evaluate PSMA binding of the unlabeled, iodinated compounds. A series of paired-label biodistribution experiments were performed to compare each 211At-labeled PSMA ligand to its 131I-labeled counterpart in mice bearing subcutaneous PC3 PSMA+ PIP xenografts. RESULTS: Radiochemical yields ranged from 32% to 65% for the 211At-labeled PSMA inhibitors and were consistently lower than those obtained with the corresponding 131I-labeled analogue. Good localization in PC3 PSMA+ PIP but not control xenografts was observed for all labeled molecules studied, which exhibited a variable degree of in vivo dehalogenation as reflected by thyroid and stomach activity levels. Normal tissue uptake and in vivo stability for several of the compounds was markedly improved compared with the previously evaluated compounds, [211At]DCABzL and [*I]DCIBzL. CONCLUSIONS AND IMPLICATIONS FOR PATIENT CARE: Compared with the first generation compound [211At]DCABzL, several of the novel 211At-labeled PSMA ligands exhibited markedly improved stability in vivo and higher tumor-to-normal tissue ratios. [211At]GV-620 has the most promising characteristics and warrants further evaluation as a targeted radiotherapeutic for prostate cancer.

Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent.

INTRODUCTION: As a consequence of their small size, high stability and high affinity, single domain antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation. METHODS: Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([131I]MEGMB) and its N-succinimidyl ester analogue, iso-[125I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts. RESULTS: [131I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and Kd = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [131I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso-[125I]SGMIB-5F7, while higher retention was seen for iso-[125I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [131I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso-[125I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [131I]MEGMIB-5F7GGC. CONCLUSION: [131I]MEGMIB-5F7GGC offers similar tumor targeting as iso-[125I]SGMIB-5F7 but with generally lower normal tissue uptake. ADVANCES IN KNOWLEDGE AND IMPLICATION FOR PATIENT CARE: The site specific nature of the [131I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling.

Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative.

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½â€¯= 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel's Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [18F]AlF-NOTA moiety with [18F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[18F]fluoronicotinyl-PEG4-methyltetrazine to provide [18F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([18F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) and immunoreactive fraction of [18F]FN-GK-5F7 were 99%, 5.4 ±â€¯0.7 nM and 72.5 ±â€¯4.3%, respectively. Tumor uptake of [18F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ±â€¯1.2% ID/g and 3.4 ±â€¯1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[125I]SGMIB-5F7 (6.9 ±â€¯1.9 %ID/g and 8.7 ±â€¯3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [18F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[125I]SGMIB-5F7 as well as those observed for the 18F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [18F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [18F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.

Labeling Monoclonal Antibody with α-emitting 211At at High Activity Levels via a Tin Precursor.

Background: In a previous clinical study, the authors evaluated the potential of antitenascin C monoclonal antibody (mAb) 81C6 labeled with 211At via the prosthetic agent N-succinimidyl 3-[211At]astatobenzoate (SAB) for the treatment of primary brain tumors. Although encouraging results were obtained, labeling chemistry failed while attempting to escalate the dose to 370 MBq. The goal of the current study was to develop a revised procedure less susceptible to radiolysis-mediated effects on 211At labeling that would be suitable for use at higher activity levels of this α-emitter. Materials and Methods: Addition of N-chlorosuccinimide to the methanol used to remove the 211At from the cryotrap after bismuth target distillation was done to thwart radiolytic decomposition of reactive 211At and the tin precursor. A series of 11 reactions were performed to produce SAB at initial 211At activity levels of 0.31-2.74 GBq from 50 µg of N-succinimidyl 3-trimethylstannylbenzoate (Me-STB), which was then reacted with murine 81C6 mAb without purification of the SAB intermediate. Radiochemical purity, immunoreactive fraction, sterility, and apyrogenicity of the 211At-labeled 81C6 preparations were evaluated. Results: Murine 81C6 mAb was successfully labeled with 211At using these revised procedures with improved radiochemical yields and decreased overall synthesis time compared with the original clinical labeling procedure. Conclusions: With 2.74 GBq of 211At, it was possible to produce 1.0 GBq of 211At-labeled 81C6 with an immunoreactive fraction of 92%. These revised procedures permit production of 211At-labeled mAbs suitable for use at clinically relevant activity levels.

Switch-on effect on conformation-specific arylamine-DNA adduct by cyclometalated Ir(III) complexes.

Arylamines are known to form covalent-DNA adducts upon metabolic activation. These covalent adducts adopt different conformational attributes, viz., major groove (B), stacked (S), and minor groove (W), and lead to different types of mutations. The conformation depends on the flanking and next flanking bases at the 3' position of the adduct. Early detection of these conformations by simple probes is an ideal and challenging task. Here, we have reported two Ir(III)-based cyclometalated complexes, viz., [Ir(ppy)2(imiphen)]+ (1) (ppy: 2-phenylpyridine; imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(furphen)]+ (2) (furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its interaction with N-acetyl-2-aminofluorene-dG (AAF-dG). The sequences used in this work are NarI sequence (-CG1G2CG3CX-) in which Gs are modified with AAF and X is either C or T. Luminescence studies reveal that the Ir(III) complexes bind to AAF-dG adduct with high specificity toward G1 and G3 compared to G2 and unmodified control. The selectivity also depends on the next flanking base as cytosine favors G3AAF, while thymine favors G1AAF in complex 1 and vice versa for complex 2. The quenching studies confirm that Ir(III) complexes bind with AAF-dG sequences through the minor groove. The outcome of this work reveals that the switch-on effect by the complexes can be utilized for determining the conformational heterogeneity of the adduct and also for similar covalent-DNA adducts.

Selective Recognition of Conformation-Specific Arylamine-DNA Adduct in Frameshift Model by [Ru(phen)2(dppz)]2.

Arylamine modification of guanine base at the G3 position in the NarI sequence (-G1G2CG3CC-) causes a frameshift mutation. Polymerase and 19F NMR studies have shown that the next flanking base at the 3' position to the G3 adduct modulates the mutational outcome because of its different conformations. Here, we have studied the interaction of the 16-mer NarI sequence (5'-CTCTCG1G2CG3CXATCAC-3') (G3 = N-acetyl-2-aminofluorene (AAF)-dG and X is either C or T) with [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2',3'-c]phenazine). Interaction studies between isomers of Ru(II) and two oligonucleotide models, viz., (a) full duplex, and (b) slipped mutagenic intermediate (SMI), have been carried out. Luminescence studies reveal that the sensitivity of Ru(II) with an adduct increases 2- to 3-fold compared to that of control in full duplex. In SMI, the sensitivity of Ru(II) varies with the next flanking base and in the order of -GAAFCC > -GAAFCT. Microscale thermophoretic data reveal that in full duplex Λ-Ru binds to -GAAFCT- by 13- and 4-fold stronger than its control and -GAAFCC-, respectively. In SMI, Δ-Ru binds to -GAAFCC- (41% stacked (S) conformer) by 3-fold while -GAAFCT- (86% major groove (B) conformer) weakens the binding of Λ-Ru by 250-fold compared to the control. The results presented here reveal that the binding of Ru(II) not only depends on conformations of the AAF-dG adduct but also is isomer-centric and might be helpful in determining the conformational heterogeneity of other covalent aryl/heterocyclic amine-DNA adducts.

Observations on the Effects of Residualization and Dehalogenation on the Utility of N-Succinimidyl Ester Acylation Agents for Radioiodination of the Internalizing Antibody Trastuzumab.

Trastuzumab is an antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers. Since trastuzumab is an internalizing antibody, two factors could play an important role in achieving high uptake and prolonged retention of radioactivity in HER2-positive tumors after radioiodination-residualizing capacity after receptor-mediated internalization and susceptibility to dehalogenation. To evaluate the contribution of these two factors, trastuzumab was radiolabeled using the residualizing reagent N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB) and the nonresidualizing reagent N-succinimidyl-3-[*I]iodobenzoate ([*I]SIB), both of which are highly dehalogenation-resistant. Paired-label uptake and intracellular retention of [125I]SGMIB-trastuzumab and [131I]SIB-trastuzumab was compared on HER2-expressing BT474 human breast carcinoma cells. Tumor uptake and normal tissue distribution characteristics for the two labeled conjugates were assessed in mice bearing BT474M1 xenografts. The internalization and intracellular retention of initially-bound radioactivity in BT474 cells was similar for the two labeled conjugates up to 4 h, but were significantly higher for [125I]SGMIB-trastuzumab at 6 and 24 h. Similarly, [*I]SGMIB labeling resulted in significantly higher uptake and retention of radioactivity in BT474M1 xenografts at all studied time points. Moreover, tumor-to-tissue ratios for [125I]SGMIB-trastuzumab were consistently higher than those for [131I]SIB-trastuzumab starting at 12 h postinjection. Thus, optimal targeting of HER2-positive breast cancers with a radioiodinated trastuzumab conjugate requires an acylation agent that imparts residualizing capacity in addition to high stability towards dehalogenation in vivo.

Selective recognition of DNA defects by cyclometalated Ir(iii) complexes.

Selective detection of DNA defects is an attractive approach for early detection of various tumors and diseases. Ru(ii), Rh(iii) and hybrid complexes have been reported in this regard. Here, we present three heteroleptic cyclometalated Ir(iii) complexes [Ir(phpy)2(imiphen)]+(Ir-1), [Ir(phpy)2(furphen)]+(Ir-2) and [Ir(phpy)2(faqphen)]+(Ir-3) where imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline; furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline and faqphen: 2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)anthracene-9,10-dione have been synthesized and characterized by analytical techniques. All three complexes recognize DNA defects with high selectivity as Ir-1 recognizes the abasic site opposite T and CA mismatch, Ir-2 recognizes Ab : G and Ir-3 recognizes the TT mismatch preferably. The results from this study further emphasize that the development of new luminescent probes requires fine-tuning of the ligand structure and the choice of the metal center to improve selectivity in recognition of DNA defects.