68Ga-Labeling: Laying the Foundation for an Anti-Radiolytic Formulation for NOTA-sdAb PET Tracers.

During the preparation of [68Ga]Ga-NOTA-sdAb at high activity, degradation of the tracers was observed, impacting the radiochemical purity (RCP). Increasing starting activities in radiolabelings is often paired with increased degradation of the tracer due to the formation of free radical species, a process known as radiolysis. Radical scavengers and antioxidants can act as radioprotectant due to their fast interaction with formed radicals and can therefore reduce the degree of radiolysis. This study aims to optimize a formulation to prevent radiolysis during the labeling of NOTA derivatized single domain antibody (sdAbs) with 68Ga. Gentisic acid, ascorbic acid, ethanol and polyvinylpyrrolidone were tested individually or in combination to find an optimal mix able to prevent radiolysis without adversely influencing the radiochemical purity (RCP) or the functionality of the tracer. RCP and degree of radiolysis were assessed via thin layer chromatography and size exclusion chromatography for up to three hours after radiolabeling. Individually, the radioprotectants showed insufficient efficacy in reducing radiolysis when using high activities of 68Ga, while being limited in amount due to negative impact on radiolabeling of the tracer. A combination of 20% ethanol (VEtOH/VBuffer%) and 5 mg ascorbic acid proved successful in preventing radiolysis during labeling with starting activities up to 1-1.2 GBq of 68Ga, and is able to keep the tracer stable for up to at least 3 h after labeling at room temperature. The prevention of radiolysis by the combination of ethanol and ascorbic acid potentially allows radiolabeling compatibility of NOTA-sdAbs with all currently available 68Ge/68Ga generators. Additionally, a design is proposed to allow the incorporation of the radioprotectant in an ongoing diagnostic kit development for 68Ga labeling of NOTA-sdAbs.

Lyophilization of NOTA-sdAbs: First step towards a cold diagnostic kit for 68Ga-labeling.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging. METHODS: NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2-8 °C by evaluating the precursor integrity, aggregation, functionality and 68Ga-labeling efficiency. A comparative biodistribution study (lyophilized vs non-lyophilized precursor) was conducted in wild type mice (n = 3) and in tumor bearing mice (n = 6). RESULTS: The lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2-8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity after 68Ga-labeling is consistent over a period of 12 months (RCP ≥ 95% at T12m). In vivo biodistribution analyses show a typical [68Ga]Ga-NOTA-anti-HER2 sdAb distribution profile and a comparable tumor uptake for the lyophilized compound vs non-lyophilized (5.5% vs 5.7 %IA/g, respectively). In vitro results of lyophilized NOTA-anti-MMR precursor indicates stability for up to 18 months, while in vivo data show a comparable tumor uptake (2.5% vs 2.8 %IA/g, respectively) and no significant difference in kidney retention (49.4% vs 47.5 %IA/g, respectively). CONCLUSION: A formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2-8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of 68Ga-sdAb-based radiopharmaceuticals.

Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality.

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages.

PURPOSE: Macrophage mannose receptor (MMR, CD206) expressing tumor-associated macrophages (TAM) are protumorigenic and was reported to negatively impact therapy responsiveness and is associated with higher chances of tumor relapse following multiple treatment regimens in preclinical tumor models. Since the distribution of immune cells within the tumor is often heterogeneous, sampling "errors" using tissue biopsies will occur. In order to overcome this limitation, we propose positron emission tomography (PET)/X-ray computed tomography (CT) imaging using 68Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM. PROCEDURES: Cross-reactive anti-MMR-sdAb was produced according to good manufacturing practice (GMP) and conjugated to p-SCN-Bn-NOTA bifunctional chelator for 68Ga-labeling. Biodistribution and PET/CT studies were performed in wild-type and MMR-deficient 3LL-R tumor-bearing mice. Biodistribution data obtained in mice were extrapolated to calculate radiation dose estimates for the human adult using OLINDA software. A 7-day repeated dose toxicity study for NOTA-anti-MMR-sdAb was performed in healthy mice up to a dose of 1.68 mg/kg. RESULTS: [68Ga]Ga-NOTA-anti-MMR-sdAb was obtained with 76 ± 2 % radiochemical yield, 99 ± 1 % radiochemical purity, and apparent molar activity of 57 ± 11 GBq/µmol. In vivo biodistribution analysis showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor, with tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The calculated effective dose was 0.027 mSv/MBq and 0.034 mSv/MBq for male and female, respectively, which means that a proposed dose of 185 MBq in humans would yield a radiation dose of 5.0 and 6.3 mSv to male and female patients, respectively. In the toxicity study, no adverse effects were observed. CONCLUSIONS: Preclinical validation of [68Ga]Ga-NOTA-anti-MMR-sdAb showed high specific uptake of this tracer in MMR-expressing TAM and organs, with no observed toxicity. [68Ga]Ga-NOTA-anti-MMR-sdAb is ready for a phase I clinical trial.

Evaluation of [99mTc]Radiolabeled Macrophage Mannose Receptor-Specific Nanobodies for Targeting of Atherosclerotic Lesions in Mice.

PURPOSE: Macrophage accumulation characterizes the development of atherosclerotic plaques, and the presence of certain macrophage subsets might be an indicator of plaque phenotype and (in)stability. The macrophage mannose receptor (MMR) is expressed on alternatively activated macrophages and found at sites of intraplaque hemorrhage and neovascularization. It has been proposed as target to identify vulnerable plaques. Therefore, we aimed to assess the feasibility of using anti-MMR nanobodies (Nbs) as molecular tracers for nuclear imaging in an animal model of atherosclerosis. PROCEDURE: Anti-MMR and control Nb, radiolabeled with Tc-99m, were injected in ApoE-/- and/or C57Bl/6 mice (n = 6). In vivo competition studies involving pre-injection of excess of unlabeled anti-MMR Nb (n = 3) and injection of anti-MMR Nb in MMR-/- mice (n = 3) were performed to demonstrate specificity. At 3 h p.i. radioactive uptake in organs, tissues and aorta segments were evaluated. Autoradiography and immunofluorescence were performed on aortic sections. RESULTS: Significantly higher uptake was observed in all aortic segments of ApoE-/- mice injected with anti-MMR Nb compared to control Nb (1.36 ± 0.67 vs 0.38 ± 0.13 percent of injected dose per gram (%ID/g), p ≤ 0.001). Surprisingly, high aortic uptake was also observed in C57Bl/6 mice (1.50 ± 0.43%ID/g, p ≥ 0.05 compared to ApoE-/-), while aortic uptake was reduced to background levels in the case of competition and in MMR-/- mice (0.46 ± 0.10 and 0.22 ± 0.06%ID/g, respectively; p ≤ 0.001). Therefore, expression of MMR along healthy aortas was suggested. Autoradiography showed no specific radioactive signal within atherosclerotic plaques, but rather localization of the signal along the aorta, correlating with MMR expression in perivascular tissue as demonstrated by immunofluorescence. CONCLUSIONS: No significant uptake of MMR-specific Nb could be observed in atherosclerotic lesions of ApoE-/- mice in this study. A specific perivascular signal causing a non-negligible background level was demonstrated. This observation should be considered when using MMR as a target in molecular imaging of atherosclerosis, as well as use of translational animal models with vulnerable plaques.