Sharma Sai Kiran, Glaser Jonathan M, Edwards Kimberly J, Khozeimeh Sarbisheh Elaheh, Salih Akam K, Lewis Jason S, Price Eric W
Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States.
Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N-5C9, Canada.
Bioconjug Chem. 2021 Jul 21;32(7):1177-1191. doi: 10.1021/acs.bioconjchem.0c00087. Epub 2020 Apr 1.
Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([Zr]Zr)-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 Zr-radiolabeling of antibodies and their impact on the and 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 ([Zr]Zr) 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 ()/specific activity (), immunoreactive fraction, and ultimately the 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 / (∼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 performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the 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 ([Zr]Zr)-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([Zr]Zr)-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 / (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86-93%), and favorable biodistribution profile with excellent tumor uptake.
使用去铁胺（DFO）偶联的锆 - 89（[Zr]Zr）标记抗体的免疫正电子发射断层扫描（Immuno - PET）是一种用于临床前和临床分子成像的强大工具。然而，尚未充分开展一项全面研究来评估抗体的DFO偶联和Zr放射性标记过程中涉及的变量及其对所得放射免疫缀合物的[具体内容缺失]和[具体内容缺失]行为的影响。在此，我们合成了HER2靶向抗体（Ab）曲妥珠单抗的不同DFO偶联物，命名为T5、T10、T20、T60和T200，以表示用于生物偶联的DFO的摩尔当量。接下来，我们在一系列全面的反应条件下用（[Zr]Zr）对免疫缀合物进行放射性标记，这些条件包括不同的缓冲液（PBS、螯合PBS、TRIS/HCl、HEPES；±辐射防护剂）、不同的反应体积（0.1 - 1 mL）、不同量的DFO偶联Ab（5、25、50 μg）以及放射性（0.2 - 1.0 mCi；7.4 - 37 MBq）。我们评估了这些变量对放射化学产率（RCY）、摩尔活度（[具体内容缺失]）/比活度（[具体内容缺失]）、免疫反应性分数的影响，并最终评估了曲妥珠单抗放射免疫缀合物的[具体内容缺失]生物分布概况和肿瘤靶向能力。我们发现，增加曲妥珠单抗的DFO偶联程度会提高RCY（约90%）和[具体内容缺失]/[具体内容缺失]（约194 MBq/nmol；35 mCi/mg），但会降低HER2结合亲和力（3.5× - 4.6×）以及曲妥珠单抗的免疫反应性分数至50 - 64%，这导致放射免疫缀合物的[具体内容缺失]性能显著下降。基于细胞的免疫反应性测定以及使用表面等离子体共振（SPR）的标准结合亲和力分析并未预测出最极端的T200缀合物的不良[具体内容缺失]性能。然而，基于SPR的无浓度校准分析得出了活性抗体浓度，并能预测[具体内容缺失]趋势。在HER2阳性异种移植模型中的正电子发射断层扫描（PET）成像和生物分布研究显示，注射放射免疫缀合物后120小时，（[Zr]Zr） - T5（约1.4 ± 0.5个DFO/Ab）在肿瘤中的活性浓度为38.7 ± 3.8 %ID/g，在肝脏中的活性浓度为6.3 ± 4.1 %ID/g。另一方面，（[Zr]Zr） - T200（10.9 ± 0.7个DFO/Ab）在肿瘤中的活性浓度为16.
