William J. McBride

A Novel Method of 18F Radiolabeling for PET

Small biomolecules are typically radiolabeled with 18F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein 18F is first attached to aluminum as Al18F, which is then bound to a chelate attached to a peptide, forming a stable Al18F-chelate-peptide complex in an efficient 1-pot process. Methods: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl3·6H2O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of 18F to form the Al18F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH2) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100°C for 15 min. In vitro and in vivo stability and targeting ability of the Al18F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). Results: The radiolabeled peptide was produced in 5%−20% yield with an estimated specific activity of 18,500–48,100 GBq (500–1,300 Ci)/mmol. The Al18F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al18F had higher tissue uptake, particularly in the bones, than the chelated Al18F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al18F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. Conclusion: The ability to bind highly stable Al18F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

Multifunctional Antibodies by the Dock-and-Lock Method for Improved Cancer Imaging and Therapy by Pretargeting

The Dock-and-Lock (DNL) method, which makes bioactive molecules with multivalency and multifunctionality, is a new approach to develop targeting molecules for improved cancer imaging and therapy. It involves the use of a pair of distinct protein domains involved in the natural association between cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and A-kinase anchoring proteins (AKAPs). The dimerization and docking domain found in the regulatory subunit of PKA and the anchoring domain (AD) of an interactive AKAP are each attached to a biologic entity, and the resulting derivatives, when combined, readily form a stably tethered complex of a defined composition that fully retains the functions of the individual constituents. The DNL method has generated several trivalent, bispecific, binding proteins, each consisting of 2 identical Fab fragments linked site-specifically to a different Fab fragment. For example, 2 identical Fabs reacting with carcinoembryonic antigen (CEA) are bound to a Fab reacting with a hapten peptide that bears a diagnostic or therapeutic radionuclide. Using a 2-step, pretargeting method that separates the bivalent anti-CEA antibody targeting of tumor from the delivery of the radioactive peptide that is captured by the second Fab of the tri-Fab construct, an improved method of cancer imaging and therapy has been developed and shows very sensitive and specific targeting of CEA-expressing tumors for either diagnostic imaging, such as with immunoSPECT and immunoPET, or radioimmunotherapy. Improved therapeutic efficacy is shown with pretargeting in a pancreatic cancer xenograft model given a tri-Fab to a pancreatic cancer MUC1 and the hapten peptide labeled with 90Y.

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as ∼0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by ≥40-fold (∼10:1 at 1 h, ∼100:1 at 24 h), compared to a 99mTc-labeled CEA-specific F(ab′) used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold (∼20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.

Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex

We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.

A novel facile method of labeling octreotide with (18)F-fluorine.

Several methods have been developed to label peptides with 18F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of 18F-aluminum fluoride (Al18F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH+ 1305) [IMP466]) with 18F. Methods: Octreotide was conjugated with the NOTA chelate and labeled with 18F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of 18F-IMP466 was studied in AR42J tumor–bearing mice and compared with that of 68Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. Results: IMP466 was labeled with Al18F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al18F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37°C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the 19F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of 18F-IMP466 (28.3 ± 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 ± 90), which could be blocked by an excess of unlabeled peptide (8.6 ± 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of 68Ga-IMP466 was similar to that of 18F-IMP466. 18F-IMP466 was stable in vivo, because bone uptake was only 0.4 ± 0.2 %ID/g, whereas free Al18F accumulated rapidly in the bone (36.9 ± 5.0 %ID/g at 2 h after injection). Small-animal PET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor. Conclusion: NOTA-octreotide could be labeled rapidly and efficiently with 18F using a 2-step, 1-pot method. The compound was stable in vivo and showed rapid accretion in somatostatin receptor subtype 2–expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with 18F.

Improving the Delivery of Radionuclides for Imaging and Therapy of Cancer Using Pretargeting Methods

The article reviews the background and current status of pretargeting for cancer imaging and therapy with radionuclides. Pretargeting procedures were introduced ∼20 years ago as an alternative to directly radiolabeled antibodies. Because they were multistep processes, they were met with resistance but have since progressed to simple and improved procedures that could become the next generation of imaging and therapy with radionuclides. The separation of the radiolabeled compound from the antibody-targeting agent affords pretargeting procedures considerable flexibility in the radiolabeling process, providing opportunities for molecular imaging using γ- or positron-emitting radionuclides and a variety of β- and α-emitting radionuclides of therapeutic applications. Pretargeting methods improve tumor/nontumor ratios, exceeding that achieved with directly radiolabeled Fab′ fragments, particularly within just a few hours of the radionuclide injection. In addition, tumor uptake exceeds that of a Fab′ fragment by as much as 10-fold, giving pretargeting a greatly enhanced sensitivity for imaging. Advances in molecular biology have led to the development of novel binding proteins that have further improved radionuclide delivery in these systems. Studies in a variety of hematologic and solid tumor models have shown advantages of pretargeting compared with directly radiolabeled IgG for therapy, and there are several clinical studies under way that are also showing promising results. Thus, the next generation of targeting agents will likely employ pretargeting approaches to optimize radionuclide delivery for a wide range of applications.

Metastatic Human Colonic Carcinoma: Molecular Imaging with Pretargeted SPECT and PET in a Mouse Model

PURPOSE To prospectively determine if a bispecific monoclonal antibody (MoAb) pretargeting method with a radiolabeled hapten peptide can depict small (<0.3 mm in diameter) microdisseminated human colon cancer colonies in the lungs of nude mice. MATERIALS AND METHODS Animal studies were approved in advance by animal care and use committees. Animals injected intravenously with a human colon cancer cell line to establish microdisseminated colonies in the lungs were pretargeted with TF2--a recombinant, humanized, anti-carcinoembryonic antigen (CEA) and anti-histamine-succinyl-glycine (HSG) bispecific MoAb; 21 hours later, a radiolabeled HSG peptide was given. Imaging and necropsy data for tumor-bearing animals given the anti-CEA bispecific MoAb (n = 38, all studies) were compared with those of animals given fluorine 18 ((18)F) fluorodeoxyglucose (FDG) (n = 15, all studies), peptide alone (n = 20, all studies), or an irrelevant anti-CD22 bispecific MoAb (n = 12, all studies). Uptake of these agents in the lungs of non-tumor-bearing animals enabled assessment of specificity (n = 15, 4, and 6 for TF2 pretarget, hapten peptide alone, and (18)F-FDG, respectively). RESULTS TF2-pretargeting helped localize tumors in the lungs within 1.5 hours of the radiolabeled HSG peptide injection, while the peptide alone, irrelevant bispecific MoAb pretargeted peptide, and (18)F-FDG failed. Necropsy data indicated that the signal in tumor-bearing lungs was five times higher than in blood within 1.5 hours, increasing to 50 times higher by 24 hours. Peptide uptake in tumor-bearing lungs pretargeted with TF2 was nine times higher than in non-tumor-bearing lungs, while it was only 1.5-fold higher with (18)F-FDG or the peptide alone. Micro-positron emission tomographic (PET) images showed discrete uptake in individual metastatic tumor colonies; autoradiographic data demonstrated selective targeting within the lungs, including metastases less than 0.3 mm in diameter. CONCLUSION Bispecific antibody pretargeting is highly specific for imaging micrometastatic disease and may thus provide a complementary method to (18)F-FDG at clinical examination.

High-Yielding Aqueous 18F-Labeling of Peptides via Al18F Chelation

The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, and its ability to form stable Al(18)F chelates were investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF(3). X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55-89%) aqueous (18)F-labeling was achieved in 10-15 min with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al(18)F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.

Pretargeted Immuno–Positron Emission Tomography Imaging of Carcinoembryonic Antigen–Expressing Tumors with a Bispecific Antibody and a 68Ga- and 18F-Labeled Hapten Peptide in Mice with Human Tumor Xenografts

18F-Fluorodeoxyglucose (18F-FDG) is the most common molecular imaging agent in oncology, with a high sensitivity and specificity for detecting several cancers. Antibodies could enhance specificity; therefore, procedures were developed for radiolabeling a small (∼1451 Da) hapten peptide with 68Ga or 18F to compare their specificity with 18F-FDG for detecting tumors using a pretargeting procedure. Mice were implanted with carcinoembryonic antigen (CEA; CEACAM5)–expressing LS174T human colonic tumors and a CEA-negative tumor, or an inflammation was induced in thigh muscle. A bispecific monoclonal anti-CEA × anti-hapten antibody was given to mice, and 16 hours later, 5 MBq of 68Ga- or 18F-labeled hapten peptides were administered intravenously. Within 1 hour, tissues showed high and specific targeting of 68Ga-IMP-288, with 10.7 ± 3.6% ID/g uptake in the tumor and very low uptake in normal tissues (e.g., tumor-to-blood ratio of 69.9 ± 32.3), in a CEA-negative tumor (0.35 ± 0.35% ID/g), and inflamed muscle (0.72 ± 0.20% ID/g). 18F-FDG localized efficiently in the tumor (7.42 ± 0.20% ID/g) but also in the inflamed muscle (4.07 ± 1.13% ID/g) and in several normal tissues; thus, pretargeted 68Ga-IMP-288 provided better specificity and sensitivity. Positron emission tomography (PET)/computed tomography images reinforced the improved specificity of the pretargeting method. 18F-labeled IMP-449 distributed similarly in the tumor and normal tissues as the 68Ga-labeled IMP-288, indicating that either radiolabeled hapten peptide could be used. Thus, pretargeted immuno-PET does exceptionally well with short-lived radionuclides and is a highly sensitive procedure that is more specific than 18F-FDG-PET. Mol Cancer Ther; 9(4); 1019–27. ©2010 AACR.

A Novel Bispecific, Trivalent Antibody Construct for Targeting Pancreatic Carcinoma

Preclinical and clinical studies have demonstrated the application of radiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy of pancreatic carcinoma. We have now examined the ability of a novel PAM4-based, bispecific monoclonal antibody (mAb) construct, TF10, to pretarget a radiolabeled peptide for improved imaging and therapy. TF10 is a humanized, bispecific mAb, divalent for mAb-PAM4 and monovalent for mAb-679, reactive against the histamine-succinyl-glycine hapten. Biodistribution studies and nuclear imaging of the radiolabeled TF10 and/or TF10-pretargeted hapten-peptide (IMP-288) were conducted in nude mice bearing CaPan1 human pancreatic cancer xenografts. (125)I-TF10 cleared rapidly from the blood, with levels decreasing to <1% injected dose per gram (ID/g) by 16 hours. Tumor uptake was 3.47 +/- 0.66% ID/g at this time point with no accumulation in any normal tissue. To show the utility of the pretargeting approach, (111)In-IMP-288 was administered 16 hours after TF10. At 3 hours postadministration of radiolabeled peptide, imaging showed intense uptake within the tumors and no evidence of accretion in any normal tissue. No targeting was observed in animals given only the (111)In-peptide. Tumor uptake of the TF10-pretargeted (111)In-IMP-288 was 24.3 +/- 1.7% ID/g, whereas for (111)In-IMP-288 alone it was only 0.12 +/- 0.002% ID/g at 16 hours. Tumor/blood ratios were significantly greater for the pretargeting group ( approximately 1,000:1 at 3 hours) compared with (111)In-PAM4-IgG ( approximately 5:1 at 24 hours; P < 0.0003). Radiation dose estimates suggested that TF10/(90)Y-peptide pretargeting would provide a greater antitumor effect than (90)Y-PAM4-IgG. Thus, the results suggest that TF10 pretargeting may provide improved imaging for early detection, diagnosis, and treatment of pancreatic cancer as compared with directly radiolabeled PAM4-IgG.