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.

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.

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.

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 Single Domain Antibody Fragments with Fluorine-18 Using 2,3,5,6-Tetrafluorophenyl 6-[18F]Fluoronicotinate Resulting in High Tumor-to-Kidney Ratios.

ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate ( iso-[125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was comparable to those for the coadministered 125I-labeled sdAb, renal uptake of the 18F-labeled sdAbs was substantially lower. In microPET images, the tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues, except the bladder. These results indicate that the [18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

Brush border enzyme-cleavable linkers: Evaluation for reducing renal uptake of radiolabeled prostate-specific membrane antigen inhibitors.

INTRODUCTION: Radiolabeled, low-molecular-weight prostate-specific membrane antigen (PSMA) inhibitors based on the Glu-ureido pharmacophore show promise for the detection and treatment of castration-resistant prostate cancer; however, high renal retention of activity, related in part to overexpression of PSMA in kidneys can be problematic. The goal of the current study was to investigate the use of brush border enzyme-cleavable linkers as a strategy for reducing kidney activity levels from radiolabeled PSMA inhibitors. METHODS: PSMA-769 (6), a derivative of the prototypical PSMA inhibitor (((S)­1­carboxy­5­(4­iodobenzamido)pentyl)carbamoyl)glutamate (12) modified to contain a Gly-Tyr linker, and its protected tin precursor (11) were synthesized starting from the basic pharmacophore molecule Lys-urea-Glu. An analogue of 6 containing d­tyrosine in lieu of l­tyrosine (PSMA-769-d-tyrosine) and the corresponding tin precursor (d-11) also were synthesized. Both radioiodinated and 211At-labeled 6 were synthesized by radiohalogenation of 11 and deprotection in situ. Similarly, radioiodinated d-6 was synthesized from d-11. Paired label biodistribution of [125I]12 and [131I]6 was performed in normal mice and in SCID mice bearing both PC3 PIP (PSMA+) and PC3 flu (PSMA-) subcutaneous prostate carcinoma xenografts. The biodistribution of [131I]6 and [211At]6 was also evaluated in this tumor model. Biodistribution of the two radioiodinated diastereomers of 6 was evaluated in normal mice and urine samples were analyzed for the presence of 4­iodohippuric acid. RESULTS: Compounds [131I]6 and [211At]6 were synthesized from 11 in overall radiochemical yields of 32.5 ±â€¯0.1% (n = 4) and 22% (n = 1), respectively; radiochemical purity was >95%. In normal mice, renal uptake of [131I]6 was 1.4-, 2.8- and 161-fold lower than that seen for co-injected [125I]12 at 1 h, 4 h and 21 h, respectively. In tumor-bearing mice, kidney uptake of [131I]6 was similar to that for [125I]12 (P > 0.05) at 1 h and 4 h but was 6- to 7-fold lower at 21 h; however, [131I]6 uptake in PC3 PIP tumors was also lower than that seen for [125I]12 at 21 h (12.6 ±â€¯3.4%ID/g vs. 36.8 ±â€¯12.4%ID/g). Uptake of [211At]PSMA-769 in PC3 PIP tumors was slightly higher than that seen for [131I]PSMA-769 at 4 h (9.6 ±â€¯1.6%ID/g versus 7.8 ±â€¯1.6%ID/g; P = 0.002); its uptake in a number of normal tissues also was higher. In normal mice, kidney uptake of [125I]PSMA-769 at 4 h was about 73% of that seen for [131I]PSMA-769-d-tyrosine. Activity in the urine of mice receiving [125I]PSMA-769 contained mainly 4­[125I]iodohippuric acid while unmetabolized intact molecule was present in the case of [125I]PSMA-769-d-tyrosine. CONCLUSION: Use of this brush border enzyme-cleavable linker reduced kidney uptake and resulted in improved tumor:kidney uptake ratios. Although further structural improvements are needed, this linker approach might be useful as a component in strategies for reducing renal uptake of radiolabeled PSMA inhibitors.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

PURPOSE: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. PROCEDURES: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. RESULTS: [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. CONCLUSIONS: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Synthesis and Preliminary Evaluation of 5-[18F]fluoroleucine.

BACKGROUND: Amino acid transporters, such as LAT1, are overexpressed in aggressive prostate and breast carcinomas, directly influencing pathways of growth and proliferation. OBJECTIVE: The purpose of this study was to synthesize and characterize a novel 18F labeled leucine analog, 5-[18F]fluoroleucine, as a potential imaging agent for aggressive tumors which may not be amenable to imaging by FDG PET. METHODS: 5-fluoroleucine was synthesized and characterized, and its 18F-labeled analog was synthesized from a mesylate precursor. First, breast cancer cell line assays were performed to evaluate uptake of 3H- or 14C-labeled L-leucine and other essential amino acids. Both L-leucine and 5- [18F]fluoroleucine were tested for uptake and accumulation over time, and for uptake via LAT1. Biodistribution studies were performed to estimate radiation dosimetry for human studies. Small animal PET / CT studies of a breast cancer were performed to evaluate in vivo 5-[18F]fluoroleucine tumor uptake. RESULTS: Breast cancer cell lines showed increasing high net accumulation of L-[14C]leucine. Both L-leucine and 5-[18F]fluoroleucine showed increasing uptake over time in in vitro tumor cell assays, and uptake was also shown to occur via LAT1. The biodistribution study of 5-[18F]fluoroleucine showed rapid renal excretion, no significant in vivo metabolism, and acceptable dosimetry for use in humans. In vivo small animal PET / CT imaging of a breast cancer xenograft showed uptake of 5- [18F]fluoroleucine in the tumor, which progressively increased over time. CONCLUSION: 5-[18F]fluoroleucine is a leucine analog which may be useful in identifying tumors with high or upregulated expression of amino acid transporters, providing additional information that may not be provided by FDG PET.

Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET.

UNLABELLED: The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. Immuno-PET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor-targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; ∼13 kDa) after (18)F labeling by 2 methods. METHODS: The 5F7 Nanobody was labeled with (18)F using the novel residualizing label N-succinimidyl 3-((4-(4-(18)F-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ((18)F-SFBTMGMB; (18)F-RL-I) and also via the most commonly used (18)F protein-labeling prosthetic agent N-succinimidyl 3-(18)F-fluorobenzoate ((18)F-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-(125)I-iodobenzoate ((125)I-SGMIB). Paired-label ((18)F/(125)I) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Small-animal PET/CT imaging of 5F7 Nanobody labeled using (18)F-RL-I also was performed. RESULTS: Internalization assays indicated that intracellularly retained radioactivity for (18)F-RL-I-5F7 was similar to that for coincubated (125)I-SGMIB-5F7, whereas that for (18)F-SFB-5F7 was lower than coincubated (125)I-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of (18)F-RL-I-5F7 was 28.97 ± 3.88 percentage injected dose per gram of tissue (%ID/g) at 1 h and 36.28 ± 14.10 %ID/g at 2 h, reduced by more than 90% on blocking with trastuzumab, indicating HER2 specificity of uptake, and was also 26%-28% higher (P < 0.05) than that of (18)F-SFB-5F7. At 2 h, the tumor-to-blood ratio for (18)F-RL-I-5F7 (47.4 ± 13.1) was significantly higher (P < 0.05) than for (18)F-SFB-5F7 (25.4 ± 10.3); however, kidney uptake was 28-36-fold higher for (18)F-RL-I-5F7. CONCLUSION: (18)F-RL-I-5F7 is a promising tracer for evaluating HER2 status by immuno-PET; however, in settings in which renal background is problematic, strategies for reducing its kidney uptake may be needed.

N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([(18)F]SFBTMGMB): a residualizing label for (18)F-labeling of internalizing biomolecules.

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the (18)F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([(18)F]SFBTMGMB-Boc2) was synthesized by a click reaction of an azide precursor and [(18)F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with (18)F using [(18)F]SFBTMGMB ([(18)F]RL-I), obtained by the deprotection of [(18)F]SFBTMGMB-Boc2, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62-80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with (18)F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

Synthesis and evaluation of 4-[18F]fluoropropoxy-3-iodobenzylguanidine ([18F]FPOIBG): A novel 18F-labeled analogue of MIBG.

INTRODUCTION: Radioiodinated meta-iodobenzylguanidine (MIBG), a norepinephrine transporter (NET) substrate, has been extensively used as an imaging agent to study the pathophysiology of the heart and for the diagnosis and treatment of neuroendocrine tumors. The goal of this study was to develop an (18)F-labeled analogue of MIBG that like MIBG itself could be synthesized in a single radiochemical step. Towards this end, we designed 4-fluoropropoxy-3-iodobenzylguanidine (FPOIBG). METHODS: Standards of FPOIBG and 4-fluoropropoxy-3-bromobenzylguanidine (FPOBBG) as well as their tosylate precursors for labeling with (18)F, and a tin precursor for the preparation of radioiodinated FPOIBG were synthesized. Radiolabeled derivatives were synthesized by nucleophilic substitution and electrophilic iododestannylation from the corresponding precursors. Labeled compounds were evaluated for NET transporter recognition in in vitro assays using three NET-expressing cell lines and in biodistribution experiments in normal mice, with all studies performed in a paired-label format. Competitive inhibition of [(125)I]MIBG uptake by unlabeled benzylguanidine compounds was performed in UVW-NAT cell line to determine IC50 values. RESULTS: [(18)F]FPOIBG was synthesized from the corresponding tosylate precursor in 5.2 ± 0.5% (n = 6) overall radiochemical yields starting with aqueous fluoride in about 105 min. In a paired-label in vitro assay, the uptake of [(18)F]FPOIBG at 2h was 10.2 ± 1.5%, 39.6 ± 13.4%, and 13.3 ± 2.5%, in NET-expressing SK-N-SH, UVW-NAT, and SK-N-BE(2c) cells, respectively, while these values for [(125)I]MIBG were 57.3 ± 8.1%, 82.7 ± 8.9%, and 66.3 ± 3.6%. The specificity of uptake of both tracers was demonstrated by blocking with desipramine. The (125)I-labeled congener of FPOIBG gave similar results. On the other hand, [(18)F]FPOBBG, a compound recently reported in the literature, demonstrated much higher uptake, albeit less than that of co-incubated [(125)I]MIBG. IC50 values for FPOIBG were higher than those obtained for MIBG and FPOBBG. Unlike the case with [(18)F]FPOBBG, the heart uptake [(18)F]FPOIBG in normal mice was significantly lower than that of MIBG. CONCLUSION: Although [(18)F]FPOIBG does not appear to warrant further consideration as an (18)F-labeled MIBG analogue, analogues wherein the iodine in it is replaced with a chlorine, fluorine or hydrogen might be worth pursuing. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: An (18)F-labeled analogue of the well-known radiopharmaceutical MIBG could have significant impact, potentially improving imaging of NET related disease in cardiology and in the imaging of neuroendocrine tumors. Although (18)F-labeled analogues of MIBG have been reported including LMI1195, we undertook this work hypothesizing that based on its greater structural similarity to MIBG, FPOIBG might be a better analogue than LMI1195.

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.

INTRODUCTION: N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position. METHODS: Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed. RESULTS: When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras. CONCLUSION: Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.

Synthesis and preliminary evaluation of n.c.a. iodoquine: a novel radiotracer with high uptake in cells with high ALDH1 expression.

PURPOSE: Chloroquine has demonstrated high affinity for aldehyde dehydrogenase 1A1 (ALDH1), an enzyme expressed in the highly tumorigenic CD133+ brain tumor initiating subpopulation. The purpose of this study is to report the novel synthesis of a chloroquine analogue, n.c.a. iodoquine, and the in vitro and in vivo uptake in cells with high ALDH1 content. METHODS AND MATERIALS: Iodoquine was synthesized in novel no-carrier-added forms (n.c.a.) for both 125I and 123I. I25I IQ and 18F FDG cell uptake assays were performed in the L1210 and L1210cpa (cyclophosphamide resistant), A549, and MG456 glioblastoma cell lines. Uptake was expressed as a percent of the administered activity. 125I IQ biodistribution studies assessed organ uptake at 1, 4, and 24 hours after IV administration (n= 15 total; 5 mice/timepoint). Radiation dosimetry estimates were calculated using standard OLINDA/EXM software. In vivo imaging of 123I IQ uptake in MG456 glioblastoma mouse model (n=10) was performed with small animal high resolution micro-SPECT. Autoradiography and histology co-localized radiotracer and tumor biodistribution. Uptake in MG456 glioblastoma tumors was quantified with gamma counting. RESULTS: L1210 cpa (high ALDH1) showed significantly higher 125I IQ uptake compared to the parental L1210 (low ALDH1) for all time points through 4 hours (20.7% ± 1.4% versus 11.0% ± 0.5%; 21.3% ± 0.9% versus 11.0% ± 0.4%; 20.6% ± 0.7% versus 9.4% ± 0.3%; and 15.7% ± 0.7% versus 7.5% + 0.4% at 30 minutes, and 1, 2 and 4 hours, respectively; p < 0.001 for all time points). In the CD133+ fraction of MG456 glioblastoma cell line, IQ uptake was significantly higher compared to FDG at all time points through 4 hours (81.5% ± 0.9% versus 1.3% ± 0.1%; 88.8% ± 0.4% versus 1.3% ± 0.1%; 87.8% ± 2.1% versus 1.7% ± 0.2%; and 87.0% ± 2.4% versus 1.8% ± 0.1 at 30 minutes, and 1, 2 and 4 hours, respectively; p > 0.001 for all time points). The A549 lung cancer cell line also showed high IQ uptake through 4 hours. IQ normal biodistribution studies showed rapid renal excretion and very low normal background brain activity after IV administration. In vivo micro-SPECT images showed mild uptake in larger MG456 glioblastomas (n=6) as verified with autoradiography and histology. Gamma well counter uptake in large tumors was 2.3% ± 0.48% ID/g (n=5). CONCLUSION: Iodoquine localizes to cells with high ALDH1 content. Cell assays show high 125I IQ uptake in the MG456 cell line, and in vivo micro-SPECT imaging showed mild 123I IQ uptake in MG456 glioblastomas. Further studies are necessary to investigate 131I IQ as a potential therapeutic agent targeting the highly tumorigenic CD133+ brain tumor stem cell subpopulation.

An alternative and expedient synthesis of radioiodinated 4-iodophenylalanine.

Radiolabeled amino acids have been used extensively in oncology both as diagnostic and therapeutic agents. In our pursuit to develop radiopharmaceuticals to target breast cancer, we were interested in determining the uptake of radioiodinated 4-iodophenylalanine, among other labeled amino acids, in breast cancer cells. In this work, we have developed an alternative method for the synthesis of this agent. The novel tin precursor, (S)-tert-butyl 2-(tert-butoxycarbonylamino)-3-(4-(tributylstannyl)phenyl)propanoate (3) was synthesized from the known, corresponding iodo derivative. Initially, the labeled 4-iodophenylalanine was synthesized from the above tin precursor in two steps with radiochemical yields of 91.6 ± 2.7% and 83.7 ± 1.7% (n=5), for the radioiodination (first) and deprotection (second) step, respectively. Subsequently, it was synthesized in a single step with an average radiochemical yield of 94.8 ± 3.4% (n=5). After incubation with MCF-7 breast cancer cells for 60 min, an uptake of up to 49.0 ± 0.7% of the input dose was seen; in comparison, the uptake of [¹4C]phenylalanine under the same conditions was 55.9 ± 0.5%. Furthermore, the uptake of both tracers was inhibited to a similar degree in a concentration-dependent manner by both unlabeled phenylalanine and 4-iodophenylalanine. With [¹4C]phenylalanine as the tracer, IC50 values of 1.45 and 2.50 mM were obtained for Phe and I-Phe, respectively, and these values for [¹²5I]I-Phe inhibition were 1.3 and 1.0 mM. In conclusion, an improved and convenient method for the synthesis of no-carrier-added 4-[(⁎)I]phenylalanine was developed and the radiotracer prepared by this route demonstrated an amino acid transporter-mediated uptake in MCF-7 breast cancer cells in vitro that was comparable to that of [¹4C]phenylalanine.

Radioiodinated O(6)-Benzylguanine derivatives containing an azido function.

INTRODUCTION: Drug resistance to alkylator chemotherapy has been primarily attributed to the DNA repair protein alkylguanine-DNA alkyltransferase (AGT); thus, personalizing chemotherapy could be facilitated if tumor AGT content could be quantified prior to administering chemotherapy. We have been investigating the use of radiolabeled O(6)-benzylguanine (BG) analogues to label and quantify AGT in vivo. BG derivatives containing an azido function were sought to potentially enhance the targeting of these analogues to AGT, which is primarily present in the cell nucleus, either by conjugating them to nuclear localization sequence (NLS) peptides or by pretargeting via bio-orthogonal approaches. METHODS: Two O(6)-(3-iodobenzyl)guanine (IBG) derivatives containing an azido moiety-O(6)-(4-azidohexyloxymethyl-3-iodobenzyl)guanine (AHOMIBG) and O(6)-(4-azido-3-iodobenzyl)guanine (AIBG)--and their tin precursors were synthesized in multiple steps and the tin precursors were converted to radioiodinated AHOMIBG and AIBG, respectively. Both unlabeled and radioiodinated AHOMIBG analogues were conjugated to alkyne-derivatized NLS peptide heptynoyl-PK(3)RKV. The ability of these radioiodinated compounds to bind to AGT was determined by a trichloroacetic acid precipitation assay and gel electrophoresis/phosphor imaging. Labeling of an AGT-AIBG conjugate via Staudinger ligation using the (131)I-labeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[(131)I]iodobenzoate, also was investigated. RESULTS: [(131)I]AHOMIBG was synthesized in two steps from its tin precursor in 52.2 ± 7.5% (n = 5) radiochemical yield and conjugated to the NLS peptide via click reaction in 50.7 ± 4.9% (n = 6) yield. The protected tin precursor of AIBG was radioiodinated in an average radiochemical yield of 69.6 ± 4.5% (n = 7); deprotection of the intermediate gave [(131)I]AIBG in 17.8 ± 4.2% (n = 9) yield. While both [(131)I]AHOMIBG and its NLS conjugate bound to AGT pure protein, their potency as a substrate for AGT was substantially lower than that of [(125)I]IBG. Uptake of [(131)I]AHOMIBG-NLS conjugate in DAOY medulloblastoma cells was up to eightfold higher than that of [(125)I]IBG; however, the uptake was not changed when the cellular AGT content was first depleted with BG treatment. [(131)I]AIBG was almost equipotent as [(125)I]IBG with respect to binding to pure AGT; however, attempts to radiolabel AGT by treatment with unlabeled AIBG followed by Staudinger ligation using the radiolabeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[(131)I]iodobenzoate were not successful. CONCLUSION: Although AHOMIBG, and AIBG were synthesized successfully in both unlabeled and radioiodinated forms, the radioiodinated compounds failed to label AGT either after NLS peptide conjugation or via Staundiger ligation. Currently, other bio-orthogonal approaches are being evaluated for labeling AGT by pretargeting.

Targeting aldehyde dehydrogenase: a potential approach for cell labeling.

INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV.

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

Structure-based design of potent retinoid X receptor alpha agonists.

A series of tetrahydrobenzofuranyl and tetrahydrobenzothienyl propenoic acids that showed potent agonist activity against RXRalpha were synthesized via a structure-based design approach. Among the compounds studied, 46a,b showed not only very good potency against RXRalpha (K(i) = 6 nM) but was also found to be greater than 167-fold selective vs RARalpha (K(i) > 1000 nM). This compound profiled out as a full agonist in a cell-based transient transfection assay (EC(50) = 3 nM). The two antipodes were separated via chiral chromatography, and 46b was found to be 40-fold more potent than 46a. Interestingly, cocrystallization of 46a,b with the RXRalpha protein generated a liganded structure whereby the (S)-antipode was found in the binding pocket. Given orally in db/db mice or ZDF rats, 46a,b showed a significant glucose-lowering effect and an increase in liver mass. Triglycerides decreased significantly in db/db mice but increased in the ZDF rats. A dose-dependent decrease of nonesterified free fatty acids was seen in ZDF rats but not in db/db mice. These differences indicate a species specific effect of RXR agonists on lipid metabolism.