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 (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 ([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 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.

The inverse electron-demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO) facilitates the efficient radiosynthesis of Ac-labelled radioimmunoconjugates in a two-step method, outperforming conventional approaches based on isothiocyanate couplings.

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 6 7.8 percentage injected dose per gram [%ID/g]), whereas uptake in MKN45 xenografts (HGF-negative) was 5.0 6 1.3 %ID/g and a control of nonspecific human IgG 89 Zr-DFO-IgG in U87MG tumors was 11.5 6 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.

OBJECTIVES: The PET imaging isotope 89Zr (t1/2 = ∼3.3 d) is the most commonly used isotope with antibodies (Abs) (immuno-PET). The chelator desferrioxamine (DFO) has been conjugated to many Abs, radiolabeled with 89Zr, and imaged in many animals and humans. It is generally known that conjugating too many chelators onto Abs will decrease binding affinity and immunoreactivity. We have conjugated DFO to the HER2/neu targeting Abs trastuzumab in varying quantities, and then radiolabeled with 89Zr under a comprehensive set of conditions and evaluated their effects on radiochemical yields (RCY), specific activity (SA), immunoreactivity, and ultimately in vivo tumor targeting. We believe a comprehensive study on these variables has not been sufficiently performed and would be of substantial value to anyone attaching isotopes to antibodies. METHODS: The chelator p-SCN-Bn-DFO was conjugated to trastuzumab (5, 10, 20, 40, 60, 100, 150, 200 equivalents), purified, and the number of attached chelators determined by MALDI-TOF mass spectrometry. These different modifications of DFO-trastuzumab were then radiolabeled and RCY were monitored from 15-60 min by iTLC (25 °C and 37 °C, n = 3, 50 mM EDTA mobile phase). The immunoreactivity of these different conjugates was determined by a cell binding assay with SKOV3 cells, and binding affinity by surface plasmon resonance. This was followed by 89Zr radiolabeling with varying quantities of Abs (5, 25, 50 μg), different buffers (PBS, chelexed PBS, TRIS/HCl, HEPES), different volumes (100, 300, 1000 μL), and activities (0.2, 1.0 mCi). To test radiolysis effects, 89Zr-DFO-trastuzumab (∼200 μCi) was radiolabeled and then placed in 100 μL of different buffers (PBS, TRIS/HCl, saline, HEPES, PBS + ascorbic acid, and PBS + gentisic acid) for 48 h before determining immunoreactivity. Representative samples (5, 10, 20, and 200 equiv. DFO conjugations) were radiolabeled and injected in mice bearing subcutaneous SKOV3 xenografts (n = 4 per group), and imaged by PET at 24, 72, and 120 h p.i. with biodistributions done at 120 h. RESULTS: Regardless of the equiv. chelator used during conjugations, a maximum average number of chelates/Abs of only ∼10-12 could be attached. To achieve maximum SA, a small quantity of Abs was used (5 μg), achieving RCY of ∼50-90% and SA of ∼15-35 mCi/mg (depending on chelates/Abs). The optimal conjugation level and radiolabeling conditions were 1-3 DFO per Abs (5-10 equiv. conjugation), chelexed PBS as buffer, minimum buffer volume, and no radioprotectant (gentisic acid or ascorbic acid). The in vivo effects of these chelate conjugations were dramatic, revealing a trend where higher amounts of attached DFO correlated to lower tumor uptake and higher liver and spleen uptake. The 89Zr-DFO-Trastuzumab that was prepared by reacting 5 equiv. of chelator (∼1.4 ± 0.5 chelates/Abs) had tumor and liver uptake at 120 h p.i. of 38.7 ± 3.8 and 6.3 ± 4.1%ID/g, respectively, where the 200 equiv. conjugation (∼10.9 ± 0.7 chelates/Abs) had values of 16.2 ± 3.2 and 27.5 ± 4.1%ID/g, respectively (p < 0.05). CONCLUSIONS: Increasing the degree of DFO attachment to trastuzumab resulted in a concomitant increase in maximum SA values (up to ∼35 mCi/mg), and a decrease in immunoreactivity/binding affinity, which translated to inferior in vivo performance. Optimized radiolabeling conditions provided very high SA of 17.5 ± 2.2 mCi/mg with only ∼1.4 ± 0.5 chelates/Abs. Citation Format: Eric W. Price, Jonathan M. Glaser, Kimberly J. Edwards, Jason S. Lewis. Optimized antibody modification and radiolabeling conditions for zirconium-89 immuno-PET. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-182.