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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 [ 68 Ga]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 [ 68 Ga]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 [ 68 Ga]Ga-DOTA-(Glu) 2 -PEG 4 -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 [ 68 Ga]Ga-DOTA-PEG 4 -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 [ 68 Ga]Ga-DOTA-Glu-PEG 4 -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 [ 68 Ga]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.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([ 89 Zr]Zr 4+ )-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 89 Zr-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 ([ 89 Zr]Zr 4+ ) 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 ( A m )/specific activity ( A s ), 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 A m / A s (∼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 ([ 89 Zr]Zr 4+ )-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([ 89 Zr]Zr 4+ )-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 A m / A s (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.

Purpose: Tumor-specific molecular imaging is an important tool for assessing disease burden and treatment response. CA19.9 is an important tumor-specific marker in several malignancies, including urothelial carcinoma. [ 89 Zr]DFO-HuMab-5B1 (MVT-2163) is a CA19.9-specific antibody-based construct that has been validated in preclinical animal models of lung, colorectal, and pancreatic malignancies for positron emission tomography (PET) imaging and is currently in a phase I trial for pancreatic cancer (NCT02687230). Here, we examine whether [ 89 Zr]DFO-HuMab-5B1 may be useful in defining urothelial malignancies., Procedures: Surface expression of CA19.9 was confirmed in the human bladder cancer line HT 1197. The radioimmunoconjugate [ 89 Zr]DFO-HuMab-5B1 was injected into mice bearing HT 1197 xenografts, and followed by PET imaging, ex vivo experiments including biodistribution, histology and autoradiography, and analysis of blood samples for shed antigen levels were performed., Results: [ 89 Zr]DFO-HuMab-5B1 specifically accumulates in HT 1197 engrafted tumors when imaged with PET. Ex vivo biodistribution of organs and autoradiography of engrafted tumors confirm our construct's specific tumor binding. The target antigen CA19.9 was not found to be shed in vitro or in vivo., Conclusions: [ 89 Zr]DFO-HuMab-5B1 can be used to delineate urothelial carcinomas by PET imaging and may provide tumor-specific information prior to, during, and after systemic therapies.

The hepatocyte growth factor (HGF) binding antibody rilotumumab (AMG102) was modified for use as a 89 Zr-based immuno-PET imaging agent to noninvasively determine the local levels of HGF protein in tumors. Because recent clinical trials of HGF-targeting therapies have been largely unsuccessful in several different cancers (e.g., gastric, brain, lung), we have synthesized and validated 89 Zr-DFO-AMG102 as a companion diagnostic for improved identification and selection of patients having high local levels of HGF in tumors. To date, patient selection has not been performed using the local levels of HGF protein in tumors. Methods: The chelator p -SCN-Bn-DFO was conjugated to AMG102, radiolabeling with 89 Zr was performed in high radiochemical yields and purity (>99%), and binding affinity of the modified antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA)-type binding assay. PET imaging, biodistribution, autoradiography and immunohistochemistry, and ex vivo HGF ELISA experiments were performed on murine xenografts of U87MG (HGF-positive, MET-positive) and MKN45 (HGF-negative, MET-positive) and 4 patient-derived xenografts (MET-positive, HGF unknown). Results: Tumor uptake of 89 Zr-DFO-AMG102 at 120 h after injection in U87MG xenografts (HGF-positive) was high (36.8 ± 7.8 percentage injected dose per gram [%ID/g]), whereas uptake in MKN45 xenografts (HGF-negative) was 5.0 ± 1.3 %ID/g and a control of nonspecific human IgG 89 Zr-DFO-IgG in U87MG tumors was 11.5 ± 3.3 %ID/g, demonstrating selective uptake in HGF-positive tumors. Similar experiments performed in 4 different gastric cancer patient-derived xenograft models showed low uptake of 89 Zr-DFO-AMG102 (∼4-7 %ID/g), which corresponded with low HGF levels in these tumors (ex vivo ELISA). Autoradiography, immunohistochemical staining, and HGF ELISA assays confirmed that elevated levels of HGF protein were present only in U87MG tumors and that 89 Zr-DFO-AMG102 uptake was closely correlated with HGF protein levels in tumors. Conclusion: The new immuno-PET imaging agent 89 Zr-DFO-AMG102 was successfully synthesized, radiolabeled, and validated in vitro and in vivo to selectively accumulate in tumors with high local levels of HGF protein. These results suggest that 89 Zr-DFO-AMG102 would be a valuable companion diagnostic tool for the noninvasive selection of patients with elevated local concentrations of HGF in tumors for planning any HGF-targeted therapy, with the potential to improve clinical outcomes., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

We have investigated novel bifunctional chelate alternatives to the aminocarboxylate macrocycles NOTA (N(3)O(3)) or DOTA (N(4)O(4)) for application of radioisotopes of Ga to diagnostic nuclear medicine and have found that the linear N(4)O(2) chelate H(2)dedpa coordinates (67)Ga quantitatively to form [(67)Ga(dedpa)](+) after 10 min at RT. Concentration-dependent coordination to H(2)dedpa of either (68)Ga or (67)Ga showed quantitative conversion to the desired products with ligand concentrations as low as 10(-7) M. With (68)Ga, specific activities as high as 9.8 mCi nmol(-1) were obtained without purification. In a 2 h competition experiment against human apo-transferrin, [(67)Ga(dedpa)](+) showed no decomposition. Two bifunctional versions of H(2)dedpa are also described, and these both coordinate to (67)Ga at RT within 10 min. Complete syntheses, characterizations, labeling studies, and biodistribution profiles of the (67)Ga complexes are presented for the new platform chelates. The stability of these platform chelates is higher than that of DOTA.