Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99mTc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99mTc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Evaluation of a 68Ga-Labeled DOTA-Tetrazine as a PET Alternative to 111In-SPECT Pretargeted Imaging.

The bioorthogonal reaction between a tetrazine and strained transcyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an 111In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([111In]In-DOTA-PEG11-Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, 111In, has been replaced with the ß-emitter, 177Lu and α-emitter, 212Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the 'universal chelator', DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a 68Ga-labeled variant ([68Ga]Ga-DOTA-PEG11-Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [68Ga]Ga-DOTA-PEG11-Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [68Ga]Ga-DOTA-PEG11-Tz can serve as an alternative to [111In]In-DOTA-PEG11-Tz.

Improved radiosynthesis and preliminary in vivo evaluation of the 11C-labeled tetrazine [11C]AE-1 for pretargeted PET imaging.

Pretargeted nuclear imaging based on the ligation between tetrazines and nano-sized targeting agents functionalized with trans-cyclooctene (TCO) has recently been shown to improve both imaging contrast and dosimetry in nuclear imaging of nanomedicines. Herein, we describe the improved radiosynthesis of a 11C-labeled tetrazine ([11C]AE-1) and its preliminary evaluation in both mice and pigs. Pretargeted imaging in mice was carried out using both a new TCO-functionalized polyglutamic acid and a previously reported TCO-functionalized bisphosphonate system as targeting agents. Unfortunately, pretargeted imaging was not successful using these targeting agents in pair with [11C]AE-1. However, brain imaging in pig indicated that the tracer crossed the blood-brain-barrier. Hence, we suggest that this tetrazine scaffold could be used as a starting point for the development of pretargeted brain imaging, which has so far been a challenging task.

Technetium(I) Complexes of Bathophenanthrolinedisulfonic Acid.

Bathophenanthrolinedisulfonate (BPS) complexes of technetium(I) of the type [Tc(CO)3(BPS)(L)]n (L = imidazole derivatives) were synthesized and evaluated both in vitro and in vivo. [99mTc(CO)3(BPS)(MeIm)]- (MeIm = 1-methyl-1H-imidazole) was prepared in near-quantitative yield using a convenient two-step, one-pot labeling procedure. A targeted analogue capable of binding regions of calcium turnover associated with bone metabolism was also prepared. Here, a bisphosphonate was linked to the metal through an imidazole ligand to give [99mTc(CO)3(BPS)(ImAln)]2- (ImAln = an imidazole-alendronate ligand) in high yield. The technetium(I) complexes were stable in vitro, and in biodistribution studies, [99mTc(CO)3(BPS)(ImAln)]2- exhibited rapid clearance from nontarget tissues and significant accumulation in the shoulder (7.9 ± 0.2% ID/g) and knees (15.1 ± 0.9% ID/g) by 6 h, with the residence time in the skeleton reaching 24 h. A rhenium analogue, which is luminescent and has the same structure, was also prepared and used for fluorescence labeling of cells in vitro. The data reported demonstrate the potential of this class of compounds for use in creating isostructural optical and nuclear probes.

Imidazole-based [2 + 1] Re(I)/99mTc(I) complexes as isostructural nuclear and optical probes.

The synthesis, stability, and photophysical properties of [2 + 1] Re(I)/Tc(I) complexes derived from bipyridine and a series of imidazole derivatives were investigated as a means of identifying complexes suitable for creating targeted isostructural optical/nuclear molecular imaging probes. To prepare the desired complexes, [Re(CO)3(H2O)3]Br was combined with 2,2'-bipyridine (bipy) to give [Re(CO)3(bipy)Br], which in turn was converted to the desired complexes by treatment with functionalized imidazoles, yielding crystal structures of two new Re complexes. The corresponding (99m)Tc complexes [(99m)Tc(CO)3(bipy)(L)](+) (L = imidazole derivatives) were prepared by combining [(99m)Tc(CO)3(bipy)(H2O)]Cl with the same series of ligands and heating at 40 or 60 °C for 30 min. Quantitative transformation to the final products was confirmed in all cases by HPLC, and the nature of the complexes was verified by comparison to the authentic Re standards. Incubation in saline and plasma, and amino acid challenge experiments showed that N-substituted imidazole derivatives, bearing electron donating groups, exhibited superior stability to analogous metal complexes derived from less basic ligands. Imaging studies in mice revealed that with the appropriate choice of monodentate ligand, it is possible to prepare robust [2 + 1] Tc complexes that can be used as the basis for preparing targeted isostructural optical and nuclear probes.

Synthesis and Radiolabeling of Glu-Urea-Lys with 99mTc-Tricarbonyl-Imidazole-Bathophenanthroline Disulfonate Chelation System and Biological Evaluation as Prostate-Specific Membrane Antigen Inhibitor.

Background: The Glu-Urea-Lys (EUK) pharmacophore as prostate-specific membrane antigen (PSMA)-targeted ligand was synthesized, radiolabeled with 99mTc-tricarbonyl-imidazole-BPS chelation system, and biological activities were evaluated. The strategy [2 + 1] ligand is applied for tricarbonyl labeling. (5-imidazole-1-yl)pentanoic acid as a monodentate ligand and bathophenanthroline disulfonate (BPS) as a bidentate ligand formed a chelate system with 99mTc-tricarbonyl. EUK-pentanoic acid-imidazole and EUK were evaluated for PSMA active site using AutoDock 4 software. Materials and Methods: EUK-pentanoic acid-imidazole was synthesized in two steps. BPS was radiolabeled with 99mTc-tricarbonyl at 100°C for 30 min. The purified 99mTc(CO)3(H2O)BPS was used to radiolabel EUK-pentanoic acid-imidazole at 100°C, 30 min. Radiochemical purity, Log P, and stability studies were carried out within 24 h. Affinity of 99mTc(CO)3BPS-imidazole-EUK was performed in the saturation binding studies using LNCaP cells at 37°C for 1 h with a range of 0.001-1000 nM radiolabeled compound range. Internalization studies were performed in LNCaP cells with 1000 nM radiolabeled compound incubated for (0-2) h at 37°C. Biodistribution was studied in normal male Balb/c mice. The artificial intelligence predicts the uptake of radiolabeled compound in tumor. Results: The structures of synthesized compounds were confirmed by mass spectroscopy. Radiochemical purity, Log P, and protein binding were ≥95%, -0.2%, and 23%, respectively. The radiolabeled compound was stable in saline and human plasma within 24 h with radiochemical purity ≥90%. There was no release of 99mTc within 4 h in competition with histidine. The affinity was 82 ± 26.38 nM, and the activity increased inside the cells over time. Biodistribution studies showed radioactivity accumulation in kidneys less than 99mTc-HYNIC-PSMA. There was a moderate accumulation of radioactivity in the liver and intestine. Conclusion: Based on the results, 99mTc(CO)3BPS-imidazole-EUK can potentially be used as an imaging agent for studies at prostate bed and distal areas. The chelate system can be potentially labeled with rhenium for imaging studies (fluorescent or scintigraphy) and therapy.

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

BACKGROUND: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an 111In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the 44Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats. RESULTS: 44Sc was obtained from an in house 44Ti/44Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with 44Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The 44Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the 44Sc-labelled Tz. Biodistribution data of the 44Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g). CONCLUSIONS: We have prepared a 44Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t1/2 = 3.97 h) proved to be a suitable radioisotope for this study.

Preparation of tetrazine-containing [2 + 1] complexes of 99mTc and in vivo targeting using bioorthogonal inverse electron demand Diels-Alder chemistry.

The aim of this work was to synthesize and evaluate [2 + 1] 99mTc(i) polypyridine complexes containing tetrazines, which along with the corresponding Re(i) complexes, represent a new class of isostructural nuclear and turn-on luminescent probes that can be derivatized and targeted using bioorthogonal chemistry. To this end, [2 + 1] complexes of 99mTc(i) of the type [99mTc(CO)3(N^N)(L)] (N^N = bathophenanthroline disulfonate (BPS) or 2,2'-bipyridine (bipy)), where the monodentate ligand (L) was a tetrazine linked to the metal through an imidazole derivative, were prepared. The desired products were obtained in nearly quantitative radiochemical yield by adding [99mTc(CO)3(N^N)(OH2)]n to the imidazole-tetrazine ligand and heating at 60 °C for 30 min. Measurement of the reaction kinetics between the tetrazine and (E)-cyclooct-4-enol revealed a second-order rate constant of 8.6 × 103 M-1 s-1 at 37 °C, which is suitable for in vivo applications that require rapid coupling. Stability studies showed that the metal complexes were resistant to ligand challenge and exhibited reasonable protein binding in vitro. Biodistribution studies of the more water-soluble BPS derivative in normal mice, one hour after administration of a bisphosphonate derivative of trans-cyclooctene (TCO-BP), revealed high activity concentrations in the knee (9.3 ± 0.3 %ID g-1) and shoulder (5.3 ± 0.7 %ID g-1). Using the same pretargeting approach, SPECT/CT imaging showed that the [2 + 1] tetrazine complex localized to implanted skeletal tumors. This is the first report of the preparation of 99mTc complexes of BPS and demonstration that their tetrazine derivatives can be used to prepare targeted imaging probes by employing bioorthogonal chemistry.

A Bone-Seeking trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using 99mTc- and 177Lu-Labeled Tetrazines.

A high yield synthesis of a novel, small molecule, bisphosphonate-modified trans-cyclooctene (TCO-BP, 2) that binds to regions of active bone metabolism and captures functionalized tetrazines in vivo, via the bioorthogonal inverse electron demand Diels-Alder (IEDDA) cycloaddition, was developed. A 99mTc-labeled derivative of 2 demonstrated selective localization to shoulder and knee joints in a biodistribution study in normal mice. Compound 2 reacted rapidly with a 177Lu-labeled tetrazine in vitro, and pretargeting experiments in mice, using 2 and the 177Lu-labeled tetrazine, yielded high activity concentrations in shoulder and knee joints, with minimal uptake in other tissues. Pretargeting experiments with 2 and a novel 99mTc-labeled tetrazine also produced high activity concentrations in the knees and shoulders. Critically, both radiolabeled tetrazines showed negligible uptake in the skeleton and joints when administered in the absence of 2. Compound 2 can be utilized to target functionalized tetrazines to bone and represents a convenient reagent to test novel tetrazines for use with in vivo bioorthogonal pretargeting strategies.