Imaging of tumor angiogenesis using 99mTc-labeled human recombinant anti-ED-B fibronectin antibody fragments.

The aim of this study was to target the angiogenesis-associated extracellular matrix protein ED-B fibronectin for molecular imaging of solid tumors. Recombinant and chemically modified derivatives of the single-chain antibody fragment (scFv) L19, capable of being labeled with 99mTc, were synthesized and radiolabeled. The resulting compounds 99mTc-AP39, 99mTc-L19-His, and 99mTc-L19-Hi20 were assessed for their imaging properties in vivo. METHODS: L19 was genetically modified by inserting either the (Gly)3-Cys-Ala (AP39) or a (His)6 tag (L19-His) sequence at the C-terminal end. Chemical modifications were performed by conjugating the bifunctional chelator Hi20 (L19-Hi20) at epsilon-Lys-NH2 residues of the molecule to allow for a direct chelator-based labeling with 99mTc. Tumor-targeting, pharmacokinetic, and scintigraphic imaging properties of the radiolabeled scFvs were evaluated in nude mice bearing murine F9 teratocarcinoma. RESULTS: 99mTc labeling of the L19 derivatives yielded radiochemically pure proteins maintaining high immunoreactivity to ED-B fibronectin, as measured by affinity chromatography. Size-exclusion high-performance liquid chromatographic analysis of labeled L19 derivatives demonstrated either dimeric species (L19-His) or a mixture of predominantly associative dimeric and monomeric species (AP39, L19-Hi20). 99mTc-AP39 showed the most favorable biodistribution and imaging properties with high and fast tumor uptake (8.3 percentage injected dose per gram at 3 h after injection), rapid blood clearance and renal excretion, leading to high signal-to-noise ratios (tumor-to-blood ratio of 6.4 at 3 h after injection), and excellent planar scintigraphy in vivo. CONCLUSION: ED-B fibronectin can be efficiently targeted by 99mTc-AP39 and scintigraphically visualized in tumor-bearing mice, providing a potentially useful clinical tool for imaging of angiogenesis-associated ED-B fibronectin-expressing human tumors.     

Radioimmunotherapy of head and neck cancer xenografts using 131I-labeled antibody L19-SIP for selective targeting of tumor vasculature.

The extra domain B of fibronectin (ED-B) is a marker of tumor angiogenesis. The human monoclonal antibody (mAb) L19-SIP (approximately 80 kDa; SIP is "small immunoprotein") has been selected for targeting of ED-B. The aim of this study was to evaluate the potential of radioimmunotherapy (RIT) with L19-SIP, either alone or in combination with cetuximab, for treatment of head and neck squamous cell carcinoma (HNSCC). Combination with cetuximab was considered because this anti-EGFR (epidermal growth factor receptor) mAb has proven value for the treatment of HNSCC. METHODS: HNSCC xenograft lines FaDu and HNX-OE were evaluated for ED-B and EGFR expression. L19-SIP was radiolabeled with 2 candidate radionuclides for RIT, 177Lu and 131I (or 125I as substitute). The biodistribution of coinjected 177Lu-L19-SIP and 125I-L19-SIP was assessed in FaDu-bearing nude mice, whereas 131I-L19-SIP was evaluated in both xenograft lines. After labeling with high-dose 131I (623-789 MBq/mg), the maximum tolerated dose (MTD) was assessed. The efficacy of RIT with injected 131I-L19-SIP, either alone or in combination with unlabeled cetuximab (1 mg 2 times a week intraperitoneally for 4 wk), was evaluated in both xenograft lines. RESULTS: Xenograft lines expressed both antigens, with similar EGFR expression and the highest ED-B expression in FaDu. Radioiodinated L19-SIP performed better than 177Lu-L19-SIP and was further exploited. The biodistribution of 131I-L19-SIP was most favorable in FaDu-bearing mice, with tumor uptake values at 24, 48, and 72 h after injection of 8.6 +/- 1.6, 5.8 +/- 0.4, and 3.4 +/- 0.2 %ID/g (%ID/g is percentage injected dose per gram of tissue), respectively, and ratios of tumor to normal tissues that gradually increased in time, such as for blood from 4.4 +/- 1.8 at 24 h to 21.4 +/- 1.7 at 72 h, after injection. RIT at the MTD level of 74 MBq caused significant tumor growth delay and improved survival in both lines. Although FaDu was most sensitive for RIT, with size reduction of all tumors, HNX-OE was most sensitive for treatment with cetuximab. The best survival and cure rates were obtained, however, when RIT and cetuximab were combined. CONCLUSION: RIT with 131I-L19-SIP appeared efficacious in HNSCC xenografts. The efficacy of RIT was enhanced by combination with cetuximab, without increase of toxicity.     

Preparation and biological evaluation of 3-[(76)Br]bromo-α-methyl-L-tyrosine, a novel tyrosine analog for positron emission tomography imaging of tumors

Introduction

3-[¹⁸F]fluoro-α-methyl-l-tyrosine ([¹⁸F]FAMT) is a useful amino acid tracer for positron emission tomography (PET) imaging of malignant tumors. FAMT analogs labeled with ⁷⁶Br, a positron emitter with a long half-life (t_1/2=16.1 h), could potentially be widely used as amino acid tracers for tumor imaging. In this study, 3-[⁷⁶Br]bromo-α-methyl-l-tyrosine ([⁷⁶Br]BAMT) was designed, and its usefulness was evaluated as a novel PET tracer for imaging malignant tumors.

Methods

In this study, both [⁷⁶Br]BAMT and [⁷⁷Br]BAMT were prepared. The in vitro and in vivo stability of [⁷⁷Br]BAMT was evaluated by HPLC analysis. Cellular uptake and retention of [⁷⁷Br]BAMT and [¹⁸F]FAMT were evaluated using LS180 colon adenocarcinoma cells. Biodistribution studies were performed in normal mice and in LS180 tumor-bearing mice, and the tumors were imaged with a small-animal PET scanner.

Results

[⁷⁷Br]BAMT was stable in vitro but was catabolized after administration in mice. Cellular accumulation and retention of [⁷⁷Br]BAMT were significantly higher than those of [¹⁸F]FAMT. In biodistribution studies, the tumor accumulation of [⁷⁷Br]BAMT was higher than that of [¹⁸F]FAMT. However, some level of debromination was seen, which caused more retention of radioactivity in the blood and organs than was seen with [¹⁸F]FAMT. PET imaging with [⁷⁶Br]BAMT enabled clear visualization of the tumor, and the whole-body image using [⁷⁶Br]BAMT was similar to that using [¹⁸F]FAMT.

Conclusions

[⁷⁷Br]BAMT showed high levels of tumor accumulation, and [⁷⁶Br]BAMT enabled clear visualization of the tumor by PET imaging. Although an improvement in stability is still needed, ⁷⁶Br-labeled FAMT analogs could potentially serve as PET tracers for the imaging of malignant tumors.     

In vivo imaging of 64Cu-labeled polymer nanoparticles targeted to the lung endothelium.

Nanoparticles (NPs) targeting the intercellular adhesion molecule 1 (ICAM-1) hold promise as a mean of delivering therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases. As these new materials become available, strategies are needed to understand their behavior in vivo. We have evaluated the use of (64)Cu and PET to noninvasively image the lung uptake and distribution of NPs coated with an anti-ICAM antibody. METHODS: Model fluorescent NPs were coated with a mixture of an anti-ICAM antibody (or nonspecific IgG) and (64)Cu-DOTA-IgG (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Biodistribution and small-animal PET and CT studies were performed in healthy mice and in mice pretreated with lipopolysaccharides (LPSs). Metabolism studies were also performed to evaluate the stability of (64)Cu-labeled NPs in lungs in vivo. RESULTS: The lungs of mice administered anti-ICAM NPs labeled with (64)Cu were clearly imaged by small-animal PET 1, 4, and 24 h after administration. Both biodistribution and small-animal imaging showed a 3- to 4-fold higher uptake in the lungs of mice injected with ICAM-targeted NPs relative to that of the control group. Lung uptake was further enhanced by pretreating the mice with LPS, presumably because of ICAM-1 upregulation. However, an approximately 2-fold decrease in lung signal was observed in each experimental group over 24 h. Metabolism studies in lung tissues harvested from mice injected with (64)Cu-labeled anti-ICAM NPs showed considerable release of a small (64)Cu-radiometabolite from the NPs beginning as early as 1 h after injection. A decrease in lung fluorescence was also observed, most likely reflecting partial release of NPs from the lungs in vivo. CONCLUSION: The use of small-animal PET to track (64)Cu-labeled nanostructures in vivo shows potential as a strategy for the preclinical screening of new NP drug delivery agents targeting the lung endothelium and other tissues. Future design optimization to prolong the stability of the radiolabel in vivo will further improve this promising approach.     

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of ²¹¹At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.     

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.     

<Superscript>124</Superscript>I-L19-SIP for immuno-PET imaging of tumour vasculature and guidance of <Superscript>131</Superscript>I-L19-SIP radioimmunotherapy

Purpose  The human monoclonal antibody (MAb) fragment L19-SIP is directed against extra domain B (ED-B) of fibronectin, a marker of tumour angiogenesis. A clinical radioimmunotherapy (RIT) trial with ¹³¹I-L19-SIP was recently started. In the present study, after GMP production of ¹²⁴I and efficient production of ¹²⁴I-L19-SIP, we aimed to demonstrate the suitability of ¹²⁴I-L19-SIP immuno-PET for imaging of angiogenesis at early-stage tumour development and as a scouting procedure prior to clinical ¹³¹I-L19-SIP RIT. Methods   ¹²⁴I was produced in a GMP compliant way via ¹²⁴Te(p,n)¹²⁴I reaction and using a TERIMO™ module for radioiodine separation. L19-SIP was radioiodinated by using a modified version of the IODO-GEN method. The biodistribution of coinjected ¹²⁴I- and ¹³¹I-L19-SIP was compared in FaDu xenograft-bearing nude mice, while ¹²⁴I PET images were obtained from mice with tumours of <50 to ∼700 mm³. Results   ¹²⁴I was produced highly pure with an average yield of 15.4 ± 0.5 MBq/μAh, while separation yield was ∼90% efficient with <0.5% loss of TeO₂. Overall labelling efficiency, radiochemical purity and immunoreactive fraction were for ¹²⁴I-L19-SIP: ∼80 , 99.9 and >90%, respectively. Tumour uptake was 7.3 ± 2.1, 10.8 ± 1.5, 7.8 ± 1.4, 5.3 ± 0.6 and 3.1 ± 0.4%ID/g at 3, 6, 24, 48 and 72 h p.i., resulting in increased tumour to blood ratios ranging from 6.0 at 24 h to 45.9 at 72 h p.i.. Fully concordant labelling and biodistribution results were obtained with ¹²⁴I- and ¹³¹I-L19-SIP. Immuno-PET with ¹²⁴I-L19-SIP using a high-resolution research tomograph PET scanner revealed clear delineation of the tumours as small as 50 mm³ and no adverse uptake in other organs. Conclusions   ¹²⁴I-MAb conjugates for clinical immuno-PET can be efficiently produced. Immuno-PET with ¹²⁴I-L19-SIP appeared qualified for sensitive imaging of tumour neovasculature and for predicting ¹³¹I-L19-SIP biodistribution. Bernard M. Tijink and Lars R. Perk contributed equally to this article.     

76Br]Bromodeoxyuridine, a potential tracer for the measurement of cell proliferation by positron emission tomography, in vitro and in vivo studies in mice

5-Bromo-2'-deoxyuridine (BrUdR) labeled with 77Br and 76Br was compared with 5-iodo-2'-deoxyuridine (IUdR) labeled with 125I or 131I, first in vitro then in in vivo experiments in mice. The results showed a significantly higher incorporation of BrUdR into DNA than IUdR, which can be explained by the greater similarity (size and surface hydrophilicity of the molecules) of BrUdR to thymidine. Both tracers are dehalogenated quickly in vivo but not in vitro. Free bromide is excreted more slowly than iodide, resulting in a higher background activity level after the application of [76Br]BrUdR and compensates for the favorable DNA incorporation. 76Br has more favorable properties than 124I for imaging purposes with positron emission tomography (PET) because of a very convenient half-life (16 h vs. 4.15 days) and about double the positron yield per decay. However, the more favorable physical properties are balanced by the slower excretion and thus the estimated radiation dose is higher in the case of 76Br than 124I. Thus, both tracers, [124I]IUdR and [76Br]BrUdR are potentially suitable but not optimal to measure cell proliferation in vivo. The difference between the two tracers is small and the extrapolation from mice to human difficult, and thus it cannot be concluded if one of the tracers would be better than the other for imaging of cancer patients.     

Immuno-PET of human colon xenograft- bearing BALB/c nude mice using 124I-CDR-grafted humanized A33 monoclonal antibody.

Radiolabeling monoclonal antibodies (mAbs) allows the evaluation of biodistribution of constructs in vivo through gamma camera imaging and also permits quantitation of mAb uptake in tumors through biopsy-based counting techniques. The quantitation of radiolabeled mAb uptake in cancer patients is complicated by the attenuation of gamma emissions of routinely used isotopes (e.g., 131I and 111In) and the spatial resolution and sensitivity of gamma cameras. METHODS: We used the positron-emitting isotope 124I (half-life [T1/2] = 4.2 d) to label the recombinant humanized anti-colorectal cancer A33 antibody (huA33) and evaluated its biodistribution properties and PET imaging characteristics in BALB/c nude mice bearing SW1222 colorectal xenografts and control colon tumors. RESULTS: The immunoreactivity of radioconjugate was 78% as determined using the cell-binding Lindmo assay. The apparent association constant was found to be 2.2 x 10(9) M(-1), and the number of antibody binding sites per cell was 371,000. The radioconjugate was found to be stable in serum obtained from mice at various times after injection. Assuming a two-compartment model with a four-parameter fit of mean blood levels, the T1/2alpha was 1.5 h and the T1/2beta was 38.2 h. Excellent tumor uptake was obtained, with maximal uptake reaching 50.0 +/- 7.0 percentage injected dose per gram of tumor by 4 d after injection. Specificity of localization was shown by lack of uptake in control tumor. PET imaging detected antigen-positive tumor by 4 h after injection, and high-resolution images were obtained by 24 h after injection. CONCLUSION: In clinical trials using PET, huA33 labeled with 124I has potential for imaging and staging colon tumors and quantifying antibody uptake in colon tumors in vivo.     

PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic antigen diabody.

In this study, we investigated the 18F-labeled anti-carcinoembryonic antigen (CEA) T84.66 diabody, a genetically engineered noncovalent dimer of single-chain variable fragments, for small-animal PET imaging of CEA expression in xenograft-bearing mice. METHODS: 18F labeling of the anti-CEA T84.66 diabody (molecular mass, 55 kDa) was achieved with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The biodistribution of the 18F-fluorobenzyl-T84.66 diabody (18F-FB-T84.66 diabody) was evaluated in athymic nude mice bearing subcutaneous LS 174T human colon carcinoma and C6 rat glioma tumors. Serial small-animal PET imaging studies were performed to further evaluate in vivo targeting efficacy and pharmacokinetics. RESULTS: Radiolabeling required 35 +/- 5 (mean +/- SD) min starting from 18F-SFB, and the tracer 18F-FB-T84.66 diabody was synthesized with a specific activity of 1.83 +/- 1.71 TBq/mmol. The decay-corrected radiochemical yield was 1.40% +/- 0.16% (n = 4), and the radiochemical purity was greater than 98%. The radioimmunoreactivity was 57.1% +/- 2.0%. The 18F-FB-T84.66 diabody showed rapid and high tumor uptake and fast clearance from the circulation in the LS 174T xenograft model, as evidenced by both small-animal PET imaging and biodistribution studies. High-contrast small-animal PET images were obtained as early as 1 h after injection of the 18F-FB-T84.66 diabody, and only a background level of activity accumulation was found in CEA-negative C6 tumors. The tracer exhibited predominantly renal clearance, with some activity in the liver and spleen at early time points. CONCLUSION: The 18F-labeled diabody represents a new class of tumor-specific probes for PET that are based on targeting cell surface antigen expression. The 18F-FB-T84.66 diabody can be used for high-contrast small-animal PET imaging of CEA-positive tumor xenografts. It may be translated to the clinic for PET of CEA-positive malignancies.     

124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice.

Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. METHODS: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody. RESULTS: PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization. CONCLUSION: (124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.     

Human antibody against C domain of tenascin-C visualizes murine atherosclerotic plaques ex vivo.

Targeting proteins that are overexpressed in atherosclerotic plaques may open novel diagnostic applications. The C domain of tenascin-C is absent from normal adult tissues but can be inserted during tumor progression or tissue repair into the molecule by alternative splicing. We tested the ability of the human antibody G11, specific to this antigen, to reveal murine atherosclerotic plaques ex vivo. The antibody directed against the extra domain B of fibronectin (L19) was used as a reference. METHODS: We intravenously injected (125)I-labeled G11 or L19 antibodies into apolipoprotein E-deficient (ApoE(-/-)) mice and harvested the aortae 4 or 24 h later. En face analyses of distal aortae and longitudinal sections of the aortic arch were performed to compare antibody uptake using autoradiography with plaque staining using oil red O. Plaque macrophages were detected by immunohistochemistry (anti-CD68 staining). Biodistribution of injected antibodies was investigated in aortae and blood at 4 and 24 h. RESULTS: En face analyses revealed a significant correlation between radiolabeled G11 and fat-stained areas, increasing from 4 to 24 h, with a correlation coefficient of 0.92 (P < 0.0001) and an average signal-to-noise ratio of 104:1 at 24 h. Plaque imaging using L19 showed similar results (r = 0.86; P < 0.0001; signal-to-noise ratio, 72:1 at 24 h). Uptake of radiolabeled antibodies in histologic sections colocalized with fat staining and activated macrophages in aortic plaques. Biodistribution analyses confirmed specific accumulation in aortic plaques as well as rapid blood pool clearance of the antibodies 24 h after injection. Immunofluorescence analyses revealed increased expression of tenascin and fibronectin isoforms in macrophage-rich plaques. CONCLUSION: The antibody G11, specific to the C domain of tenascin-C, visualizes murine atherosclerotic plaques ex vivo. In conjunction with the increased expression of the C domain of tenascin-C in macrophage-rich plaques, the colocalization of G11 uptake with activated macrophages, and the favorable target-to-blood ratio at 24 h, this antibody may be useful for molecular imaging of advanced atherosclerotic plaques in the intact organism.     

Radioimaging of light chain amyloid with a fibril-reactive monoclonal antibody.

Currently, there are no available means in the United States to document objectively the location and extent of amyloid deposits in patients with systemic forms of amyloidosis. To address this limitation, we have developed a novel diagnostic strategy, namely, the use of a radiolabeled fibril-reactive murine monoclonal antibody (mAb) as an amyloid-specific imaging agent. The goal of this study was to determine the pharmacokinetics, biodistribution, and ability of this reagent to target the type of amyloid that is formed from immunoglobulin light chains, that is, AL. METHODS: Subcutaneous tumors (amyloidomas) were induced in BALB/c mice by injection of human AL fibrils. The IgG1 mAb designated 11-1F4 and an isotype-matched control antibody were radioiodinated, and the pharmacokinetics and localization of these reagents were determined from blood and tissue samples. Amyloidoma-bearing animals that received (125)I- or (124)I-labeled antibodies were imaged by whole-body small-animal SPECT/CT or small-animal PET/CT technology, respectively. RESULTS: Radioiodinated mAb 11-1F4 retained immunoreactivity, as evidenced by its subnanomolar affinity for light chains immobilized on 96-well microtiter plates and for beads conjugated with a light chain-related peptide. Additionally, after intravenous administration, the labeled reagents had the expected biologic half-life of murine IgG1, with monoexponential whole-body clearance kinetics. In the amyloidoma mouse model, (125)I-11-1F4 was predominately localized in the tumors, as demonstrated in biodistribution and autoradiographic analyses. The mean uptake of this reagent, that is, the percentage injected dose per gram of tissue, 72 h after injection was significantly higher for amyloid than for skeletal muscle, spleen, kidney, heart, liver, or other tissue samples. Notably, the accumulation within the amyloidomas of (125)I- or (124)I-11-1F4 was readily visible in the fused small-animal SPECT/CT or small-animal PET/CT images, respectively. CONCLUSION: Our studies demonstrate the amyloid-imaging capability of a radiolabeled fibril-reactive mAb and provide the basis for a clinical trial designed to determine its diagnostic potential in patients with AL amyloidosis and other systemic amyloidoses.     

Small-animal PET of melanocortin 1 receptor expression using a 18F-labeled alpha-melanocyte-stimulating hormone analog.

(18)F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, (18)F compounds have not been successfully developed for imaging the MC1R. METHODS: In this study, an alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH(2) (NAPamide), was radiolabeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The resulting radiopeptide was evaluated as a potential molecular probe for small-animal PET of melanoma and MC1R expression in melanoma xenografted mouse models. RESULTS: The binding affinity of (19)F-SFB-conjugated NAPamide, (19)F-FB-NAPamide, was determined to be 7.2 +/- 1.2 nM (mean +/- SD) using B16/F10 cells and (125)I-(Tyr(2))-[Nle(4),D-Phe(7)]-alpha-MSH [(125)I-(Tyr(2))-NDP] as a radioligand. The biodistribution of (18)F-FB-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16/F10 melanoma tumors with high expression of MC1Rs and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Biodistribution experiments showed that tumor uptake values (percentage injected dose per gram of tumor [%ID/g]) of (18)F-FB-NAPamide were 1.19 +/- 0.11 %ID/g and 0.46 +/- 0.11 %ID/g, in B16/F10 and A375M xenografted melanoma at 1 h after injection, respectively. Furthermore, the B16/F10 tumor uptake was significantly inhibited by coinjection with excess alpha-MSH peptide (P < 0.05), indicating that (18)F-FB-NAPamide specifically recognizes the MC1R in living mice. Small-animal PET of (18)F-FB-NAPamide in mice bearing B16/F10 and A375M tumors at 1 h after tail vein injection revealed good B16/F10 tumor-to-background contrast and low A375M tumor-to-background ratios. CONCLUSION: (18)F-FB-NAPamide is a promising molecular probe for alpha-MSH receptor-positive melanoma PET and warrants further study.     

Gallium-68-labeled DOTA-rhenium-cyclized alpha-melanocyte-stimulating hormone analog for imaging of malignant melanoma

INTRODUCTION: Diagnosis of malignant melanoma is critical, since a patient's prognosis is poor. Previous studies have shown that 64Cu- and 86Y-DOTA-ReCCMSH(Arg11) have the potential for early detection of malignant melanoma by exploiting the sensitivity and high resolution of positron emission tomography (PET). This encouraged us to investigate DOTA-ReCCMSH(Arg11) labeled with another beta+-emitting radionuclide, 68Ga. METHODS: DOTA-ReCCMSH(Arg11) was successfully labeled with 68Ga at pH 3.8-4 at 85 degrees C. Acute biodistribution and small-animal PET imaging studies were performed in mice bearing B16/F1 melanoma tumor. RESULTS: Biodistribution studies showed moderate receptor-mediated tumor uptake, fast nontarget organ clearance and high tumor to nontarget tissue ratios. Preadministration of d-lysine significantly reduced kidney uptake without affecting the uptake of the agent in the tumor. Small-animal PET images showed that the tumor could be clearly visualized at all time points examined (0.5-2 h) with the standardized uptake value analysis following a similar trend as the biodistribution data. CONCLUSIONS: The preliminary data obtained suggest that 68Ga-DOTA-ReCCMSH(Arg11) is a promising PET imaging agent for early detection of malignant melanoma.     

Preclinical evaluation of the penciclovir analog 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET.

The gene for herpes simplex virus thymidine kinase (HSV-tk) is widely used as a suicide gene in experimental gene therapy of cancer. 9-(4-Fluoro-3-hydroxymethylbutyl)guanine (FHBG) is an antiviral nucleoside analog that is rapidly phosphorylated by viral thymidine kinase but is a poor substrate for mammalian thymidine kinase. Recently, FHBG labeled in the 4-fluoro position with (18)F has shown promise relative to other similar compounds for imaging in vivo expression of HSV-tk using PET. In this study, we evaluated the uptake of [(18)F]FHBG in vitro and in vivo using transduced and wild-type human colon cancer cells (HT-29). We also imaged [(18)F]FHBG and measured the radioactivity concentrations of circulating [(18)F]FHBG and its metabolites in monkeys. METHODS: Sterile, pyrogen-free [(18)F]FHBG was produced routinely in good yields. Cells were transduced with the retroviral vector G1Tk1SvNa containing HSV-tk gene. In vitro uptake studies were performed by incubating cells with [(18)F]FHBG at 37 degrees C for 1 and 5 h. Biodistribution studies were performed at 2 and 5 h after injection in nude mice bearing tumors grown from wild-type or transduced cells. Sequential, whole-body PET scans of cynomolgus monkeys were obtained over a period of >2 h after intravenous injection of [(18)F]FHBG. Arterial plasma samples obtained from monkeys 15-120 min after intravenous injection were subjected to acid extraction, and the acid-soluble fractions were analyzed by high-performance liquid chromatography. RESULTS: In vitro studies showed 31 and 71 (P < 0.001) times higher uptake of the probe at 1 and 5 h, respectively, in transduced cells compared with nontransduced cells. In vivo studies in mice showed that tumor uptake of the radiotracer was 4-fold (P < 0.05) and 13-fold (P < 0.001) higher at 2 and 5 h, respectively, in tumors grown from transduced cells compared with control cells. Transduced tumor-to-normal tissue ratios ranged from 2 to 25 at 2 h and from 2 to 22 at 5 h. Recirculating labeled metabolites had only a minor effect on the biodistribution of radiolabel from [(18)F]FHBG in monkeys. CONCLUSION: These results indicate that [(18)F]FHBG may yield high-contrast PET images of HSV-tk expression in tumors and, therefore, it is a very promising radiotracer for monitoring of gene therapy of cancer with PET.     

In vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft.

Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for noninvasive in vivo VEGF visualization and quantification with the single gamma-emitting isotope 111In and the PET isotope 89Zr. METHODS: Labeling, stability, and binding studies were performed. Nude mice with a human SKOV-3 ovarian tumor xenograft were injected with 89Zr-bevacizumab, 111In-bevacizumab, or human 89Zr-IgG. Human 89Zr-IgG served as an aspecific control antibody. Small-animal PET and microCT studies were obtained at 24, 72, and 168 h after injection of 89Zr-bevacizumab and 89Zr-IgG (3.5 +/- 0.5 MBq, 100 +/- 6 microg, 0.2 mL [mean +/- SD]). Small-animal PET and microCT images were fused to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. 89Zr- and 111In-bevacizumab ex vivo biodistribution was compared at 24, 72, and 168 h after injection (2.0 +/- 0.5 MBq each, 100 +/- 4 microg in total, 0.2 mL). RESULTS: Labeling efficiencies, radiochemical purity, stability, and binding properties were optimal for the radioimmunoconjugates. Small-animal PET showed uptake in well-perfused organs at 24 h and clear tumor localization from 72 h onward. Tumor uptake determined by quantification of small-animal PET images was higher for 89Zr-bevacizumab-namely, 7.38 +/- 2.06 %ID/g compared with 3.39 +/- 1.16 %ID/g (percentage injected dose per gram) for human 89Zr-IgG (P = 0.011) at 168 h and equivalent to ex vivo biodistribution studies. Tracer uptake in other organs was seen primarily in liver and spleen. 89Zr- and 111In-bevacizumab biodistribution was comparable. CONCLUSION: Radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Noninvasive quantitative small-animal PET was similar to invasive ex vivo biodistribution. Radiolabeled bevacizumab is a new tracer for noninvasive in vivo imaging of VEGF in the tumor microenvironment.     

Synthesis and biological evaluation of a nonsteroidal bromine-76-labeled androgen receptor ligand 3-[76Br]bromo-hydroxyflutamide

INTRODUCTION: Androgen receptors (ARs) are overexpressed in normal tissues and in most primary and metastatic prostate cancers. In our efforts to develop a nonsteroidal AR-specific imaging agent, we synthesized (+/-)-3-[(76)Br]bromo-hydroxyflutamide ((76)Br-), an analog of hydroxyflutamide, the active metabolite of the AR antagonist ligand flutamide. MATERIALS AND METHODS: (76)Br- was synthesized in three steps, starting with commercially available compounds. Labeling of (76)Br- was achieved through the nucleophilic opening of an epoxide intermediate, and a labeled compound was obtained in high specific activity and good radiochemical yield. RESULTS AND DISCUSSION: (+/-)-3-Bromo-hydroxyflutamide has a significantly higher affinity for ARs compared to hydroxyflutamide, its parent compound. The androgen target-tissue uptake of (76)Br- in diethylstilbestrol-treated male rats was examined; however, AR-mediated uptake was minimal due most likely to the rapid metabolic debromination of the radiolabeled ligand. CONCLUSIONS: This study is part of our first look at a novel class of nonsteroidal AR antagonists as positron emission tomography (PET) imaging agents, which are alternatives to steroidal AR agonist-based imaging agents. Although (76)Br- has a significant affinity for ARs, it showed limited promise as a PET imaging agent because of its poor target-tissue distribution properties.     

Imaging in vivo herpes simplex virus thymidine kinase gene transfer to tumour-bearing rodents using positron emission tomography and

Radiolabelled ganciclovir analogues have shown promise as imaging agents to detect herpes simplex virus thymidine kinase (HSVtk) expression. This study evaluated the use of positron emission tomography (PET) imaging with 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG) to assess gene transfer into tumours. HSVtk-positive and HSVtk-negative cell lines were first treated in vitro with [18F]FHPG. To assess the efficacy of PET in detecting HSVtk expression following in vivo gene transfer, mice were injected intravenously with an adenovirus encoding HSVtk (Ad.HSVtk), a control vector (Ad.Bgl2) or saline. Subcutaneous human glioma xenografts were grown in mice and treated by direct injection of Ad.HSVtk or Ad.Bgl2. Imaging was performed 48 h after transduction. Similar experiments were performed using Fischer rats implanted with syngeneic tumours. The presence of the HSVtk protein was confirmed by immunohistochemistry. Biodistribution studies were also obtained in 14 naive mice. In vitro studies showed high and specific uptake of [18F]FHPG in HSVtk-positive cell lines, with an uptake ratio of up to 27:1. PET imaging and direct counting of major organs demonstrated HSVtk-specific tracer retention. In mice, HSVtk-positive tumours retained 3.4% dose/gram as compared to 0.6% for control tumours (P=0.03). They were clearly seen on the PET images as early as 100 min post injection. Similar results were obtained with syngeneic rat tumours. Biodistribution studies demonstrated the rapid distribution and clearance of the tracer in all major organs. Our results demonstrate that PET imaging of HSVtk gene transfer to tumours is feasible and is highly specific for HSVtk expression.     

Characterization of bromine-76-labelled 5-bromo-6-nitroquipazine for PET studies of the serotonin transporter.

The development of suitable radioligands for brain imaging of the serotonin transporter is of great importance for the study of depression and other affective disorders. The potent and selective serotonin transporter ligand, 5-iodo-6-nitro-2-piperazinylquinoline, has been labelled with iodine-123 and used as a radioligand for single photon emission computerized tomography. To evaluate the potential of the bromine-76-labelled analogue, 5-bromo-6-nitroquipazine, as a radioligand for positron emission tomography (PET), its brain distribution and binding characteristics were examined in rats. In vivo brain distribution and ex vivo autoradiography demonstrated that [76Br]5-bromo-6-nitroquipazine enters the brain rapidly. The regional brain distribution of [76Br]5-bromo-6-nitroquipazine was consistent with the known distribution of serotonin transporters in the midbrain, pons, thalamus, striatum, and neocortex. Specific binding was inhibited by the selective serotonin reuptake inhibitor citalopram. The peripheral metabolism in plasma was rapid, but more than 90% of the radioactivity in brain represented unchanged radioligand 2 h postinjection (p.i.). A preliminary PET study was also performed in a baboon. Following the intravenous injection of [76Br]5-bromo-6-nitroquipazine in a baboon, there was a conspicuous accumulation of radioactivity in thalamus, striatum, and pons. The radioactivity in these brain regions was 1.5 times higher than in the cerebellum at 3 h and 2.5-4 times higher at 24 h. A rapid metabolism of the radioligand in plasma was observed (38% unchanged after 5 min). The results indicate that [76Br]5-bromo-6-nitroquipazine has potential for PET imaging of the serotonin transporter.