DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals.

Zirconium-89-labeled monoclonal antibodies and other large macromolecules such as nanoparticles hold great promise as positron emission tomography imaging agents. In general, zirconium-89 is an ideal radionuclide for long-circulating vectors such as antibodies or nanoparticles. It is also a promising radionuclide for theranostic radiopharmaceuticals due to its suitable match in half-life with actinium-225, thorium-227, lutetium-177, and others. As such, demand for new and optimized bifunctional chelators for zirconium-89 continues to grow. Herein, we present the modular chelator DFO-Km, which is octadentate and features lysine as a modular amino acid linker. The modular amino acid linker can be changed to other natural or unnatural amino acids to access different bioconjugation chemistries, while the chelating portion is unchanged thus retaining identical metal ion coordination properties to DFO-Km. The epsilon-amine in the DFO-Km linker (lysine) was used to complete synthesis of a bifunctional derivative bearing a p-SCN-Ph moiety. The chelator DFO-Km includes a redesigned hydroxamic acid, which provides more flexibility for metal ion coordination relative to the monomer used in the previously published DFO-Em. Moreover, a set of comprehensive DFT calculations were performed to model and evaluate 16 geometric isomers of Zr-(DFO-Km), which suggested the complex would form the optimum cic-cis-trans-trans octadentate Zr(IV) coordination geometry with no aqua or hydroxide ligands present. The bifunctional derivative p-SCN-Ph-DFO-Km was compared directly with the commercially available p-SCN-Ph-DFO, and both underwent efficient conjugation to a nonspecific human serum antibody (IgG) to yield two model immunoconjugates. The behavior of [89Zr]Zr-DFO-Km-IgG was studied in healthy mice for 2 weeks and compared to an equivalent cohort injected with [89Zr]Zr-DFO-IgG as a clinical "gold standard" control. PET-CT and biodistribution results revealed higher stability of [89Zr]Zr-(DFO-Km)-IgG in vivo over [89Zr]Zr-DFO-IgG, as demonstrated by the significant reduction of zirconium-89 in the whole skeleton as visualized and quantified by PET-CT at 1, 3, 7, and 14 days post-injection. Using CT-gated regions of interest over these PET-CT images, the whole skeleton was selected and uptake values were measured at 14 days post-injection of 3.6 ± 0.9 (DFO) vs 1.9 ± 0.1 (DFO-Km) %ID/g (n = 4, * p = 0.02), which represents a ∼48% reduction in bone uptake with DFO-Km relative to DFO. Biodistribution experiments performed on these same mice following the 14 day imaging time point revealed bone (both tibia) uptake values of 3.7 ± 1.3 (DFO) vs 2.0 ± 0.6 (DFO-Km) %ID/g (n = 6, * p < 0.05), with the tibia uptake values in close agreement with whole-skeleton ROI PET-CT data. These results indicate that DFO-Km is an improved chelator for [89Zr]Zr4+ applications relative to DFO. The bifunctional chelator p-SCN-Ph-DFO-Km shows potential as a new chemical tool for creating bioconjugates using targeting vectors such as antibodies, peptides, and nanoparticles.

A Systematic Investigation into the Influence of Net Charge on the Biological Distribution of Radiometalated Peptides Using [68Ga]Ga-DOTA-TATE Derivatives.

Recently, several radiometalated peptides have been approved for clinical imaging and/or therapy (theranostics) of several types of cancer; nonetheless, the primary challenge that most of these peptides confront is significant renal uptake and retention, which is often dose limiting and can cause nephrotoxicity. In response to this, numerous methods have been employed to reduce the uptake of radiometalated peptides in the kidneys, and among these is adding a linker to modulate polarity and/or charge. To better understand the influence of net charge on the biodistribution of radiometalated peptides, we selected the clinically popular construct DOTA-TATE (NETSPOT/LUTATHERA) as a model system. We synthesized derivatives using manual solid-phase peptide synthesis methods including mechanical and ultrasonic agitation to effectively yield the gold standard DOTA-TATE and a series of derivatives with different net charges (+2, +1, 0, -1, -2). Dynamic PET imaging from 0 to 90 min in healthy female mice (CD1) revealed high accumulation and retention of activity in the kidneys for the net-neutral (0) charged [68Ga]Ga-DOTA-TATE and even higher for positively charged derivatives, whereas negatively charged derivatives exhibited low accumulation and fast renal excretion. Ex vivo biodistribution at 2 h post injection demonstrated a significant retention of [68Ga]Ga-DOTA-TATE (∼74 %ID/g) in the kidneys, which increased as the net positive charge per molecule increased to +1 and +2 (∼272 %ID/g and ∼333 %ID/g, respectively), but the -1 and -2 net charged molecules exhibited lower renal uptake (∼15 %ID/g and 16 %ID/g, respectively). Interestingly, the net -2 charged [68Ga]Ga-DOTA-(Glu)2-PEG4-TATE was stable in blood serum but had much higher healthy organ uptake (lungs, liver, spleen) than the net -1 compound, suggesting instability in vivo. Although the [68Ga]Ga-DOTA-PEG4-TATE derivative with a net charge of 0 also showed a decrease in kidney uptake, it also showed instability in blood serum and in vivo. Despite the superior pharmacokinetics of the net -1 charged [68Ga]Ga-DOTA-Glu-PEG4-TATE in healthy mice with respect to kidney uptake and overall profile, dynamic PET images and ex vivo biodistribution in male mice (NSG) bearing AR42J (SSTR2 overexpressing) subcutaneous tumor xenografts showed significantly diminished tumor uptake when compared to the gold standard [68Ga]Ga-DOTA-TATE. Taken together, these findings indicate unambiguously that kidney uptake and retention are significantly influenced by the net charge of peptide-based radiotracers. In addition, it was illustrated that the negatively charged peptides had substantially decreased kidney uptake, but in this instantiation the tumor uptake was also impaired.

Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry.

Zirconium-89 has quickly become a favorite radionuclide among academics and clinicians for nuclear imaging. This radiometal has a relatively long half-life, which matches the biological half-life of most antibodies, suitable decay properties for positron emission tomography (PET), and efficient and affordable cyclotron production and purification. The "gold standard" chelator for [89Zr]Zr4+ is desferrioxamine B (DFO), and although it has been used both preclinically and clinically for immunoPET with great success, it has revealed its suboptimal stability in vivo. DFO can only bind to [89Zr]Zr4+ through its six available coordination sites made up by three hydroxamic acid (HA) moieties, which is not sufficient to saturate the coordination sphere (CN 7-8). In this study, we have designed, synthesized, and characterized a new octadentate chelator we have called DFO-Em, which is an improved derivative of our previously published dodecadentate chelator DFO2. This octadentate DFO-Em chelator is smaller than DFO2 but still satisfies the coordination sphere of zirconium-89 and forms a highly stable radiometal-chelator complex. DFO-Em was synthesized by tethering a hydroxamic acid monomer to commercially available DFO using glutamic acid as a linker, providing an octadentate chelator built on a modular amino acid-based synthesis platform. Radiolabeling performance and radiochemical stability of DFO-Em were assessed in vitro by serum stability, ethylenediamine tetraacetic acid (EDTA), and hydroxyapatite challenges. Furthermore, [89Zr]Zr-(DFO-Em) and [89Zr]Zr-DFO were injected in healthy mice and measured in vivo by PET/CT imaging and ex vivo biodistribution. Additionally, the coordination of DFO-Em with Zr(IV) and its isomers was studied using density functional theory (DFT) calculations. The radiolabeling studies revealed that DFO-Em has a comparable radiolabeling profile to the gold standard chelator DFO. The in vitro stability evaluation showed that [89Zr]Zr-(DFO-Em) was significantly more stable than [89Zr]Zr-DFO, and in vivo both had similar clearance in healthy mice with a small decrease in tissue retention for [89Zr]Zr-(DFO-Em) at 24 h post injection. The DFT calculations also confirmed that Zr-(DFO-Em) can adopt highly stable 8-coordinate geometries, which along with NMR characterization suggest no fluxional behavior and the presence of a single isomer. The modular design of DFO-Em means that any natural or unnatural amino acid can be utilized as a linker to gain access to different chemistries (e.g., thiol, amine, carboxylic acid, azide) while retaining an identical coordination sphere to DFO-Em.

A Systematic Evaluation of Antibody Modification and 89Zr-Radiolabeling for Optimized Immuno-PET.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([89Zr]Zr4+)-labeled antibodies is a powerful tool used for preclinical and clinical molecular imaging. However, a comprehensive study evaluating the variables involved in DFO-conjugation and 89Zr-radiolabeling of antibodies and their impact on the in vitro and in vivo behavior of the resulting radioimmunoconjugates has not been adequately performed. Here, we synthesized different DFO-conjugates of the HER2-targeting antibody (Ab)-trastuzumab, dubbed T5, T10, T20, T60, and T200-to indicate the molar equivalents of DFO used for bioconjugation. Next we radiolabeled the immunoconjugates with ([89Zr]Zr4+) under a comprehensive set of reaction conditions including different buffers (PBS, chelexed-PBS, TRIS/HCl, HEPES; ± radioprotectants), different reaction volumes (0.1-1 mL), variable amounts of DFO-conjugated Ab (5, 25, 50 µg), and radioactivity (0.2-1.0 mCi; 7.4-37 MBq). We evaluated the effects of these variables on radiochemical yield (RCY), molar activity (Am)/specific activity (As), immunoreactive fraction, and ultimately the in vivo biodistribution profile and tumor targeting ability of the trastuzumab radioimmunoconjugates. We show that increasing the degree of DFO conjugation to trastuzumab increased the RCY (∼90%) and Am/As (∼194 MBq/nmol; 35 mCi/mg) but decreased the HER2-binding affinity (3.5×-4.6×) and the immunoreactive fraction of trastuzumab down to 50-64%, which translated to dramatically inferior in vivo performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor in vivo performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the in vivo trends. Positron emission tomography (PET) imaging and biodistribution studies in a HER2-positive xenograft model revealed activity concentrations of 38.7 ± 3.8 %ID/g in the tumor and 6.3 ± 4.1 %ID/g in the liver for ([89Zr]Zr4+)-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([89Zr]Zr4+)-T200 (10.9 ± 0.7 DFOs/Ab) yielded 16.2 ± 3.2 %ID/g in the tumor versus 27.5 ± 4.1 %ID/g in the liver. Collectively, our findings suggest that synthesizing trastuzumab immunoconjugates bearing 1-3 DFOs per Ab (T5 and T10) combined with radiolabeling performed in low reaction volumes using Chelex treated PBS or HEPEs without a radioprotectant provided radioimmunoconjugates having high Am/As (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86-93%), and favorable in vivo biodistribution profile with excellent tumor uptake.

Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator-Peptide Conjugates.

Peptides have been widely adopted as biological targeting vectors for applications in molecular imaging and peptide-receptor radionuclide therapy (PRRT). Somatostatin (SST) analogues such as octreotate (TATE) are exogenous ligands for somatostatin receptors (SSTRs), which are highly expressed on neuroendocrine tumors (NETs). Recently, both [68Ga]Ga-DOTA-TATE (NETSPOT) and [177Lu]Lu-DOTA-TATE (LUTATHERA) received U.S. Food and Drug Administration approval for positron emission tomography (PET) imaging and PRRT of NETs, respectively. However, to the best of our knowledge a well-described synthesis of DOTA-TATE has not been reported in the literature. Herein, we report a fully reoptimized DOTA-TATE synthesis, including the application of a simple ultrasonic bath to greatly improve yields, reduce coupling times, and decrease the amount of reagents required for each coupling step by a half. The most prevalently used cyclizing agents such as iodine, thallium(III) trifluoroacetate, hydrogen peroxide, and dimethyl sulfoxide were compared. On-resin cyclizations using mechanical agitation showed higher yields (23% and 25% using I2 and Tl(III), respectively) than off-resin (1.3% and 11% using DMSO and H2O2, respectively), and the total synthesis time of DOTA-TATE was ∼540 min excluding the cyclization step, with a total synthesis yield of ∼23%. The same manual SPPS methods/reagents were reoptimized with ultrasonic (US) agitation, resulting in an immense reduction in the total synthesis time by ∼8-fold to ∼70 min for DOTA-TATE with a higher yield (∼29% yield), and ∼13-fold to 105 min for DOTA-PEG4-TATE (∼29% yield). Also, the use of US agitation reduces the need for excess molar equivalents of the reagents to a half, which is particularly important when coupling expensive or custom-synthesized groups such as bifunctional chelators and linkers. Finally, the synthesized DOTA-TATE was successfully radiolabeled with [68Ga]Ga3+ (t1/2 = 68 min) with high radiochemical yields (30 min, 95 °C). We believe this work opens the door to the facile and low-cost synthesis of many new chelator-linker-peptide conjugates that were previously cumbersome or cost-prohibitive to produce with manual SPPS.