Light-Induced Radiosynthesis of ⁸⁹Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor

Abstract

Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for positron emission tomography (PET) or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in a number of malignancies including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce 89Zr-radiolabeled onartuzumab (a monovalent, anti-human c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous 89Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing 89Zr-oxalate, the photoactive chelate DFO-aryl azide (DFO-ArN3) and MetMAb to give 89ZrDFOazepin-onartuzumab. As a control, 89ZrDFO-Bn-NCS-onartuzumab was prepared via a conventional two-step process using pre-purified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0-72 h) was performed in female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image. Tumor specificity of 89ZrDFO-azepin-onartuzumab was assessed by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced 89ZrDFO-azepin-onartuzumab in <15 min. with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity (RCP) 90% and a molar activity (Am) of 1.5 MBq nmol-1. Reaction optimization improved the radiochemical conversion (RCC) of 89ZrDFO-azepin-onartuzumab to 56.9±4.1% (n = 3), with isolated RCYs of 41.2±10.6% (n = 3), and RCPs >90%. Conventional methods produced 89ZrDFO-Bn-NCSonartuzumab with isolated RCY >97%, RCP >97% and Am 14.0 MBq nmol-1. Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake reached 15.37±5.21 %ID g-1, 6.56±4.03 %ID g-1, respectively for 89ZrDFO-azepin-onartuzumab (n = 4), and 21.38±11.57 %ID g-1 and 18.84±6.03 %ID g-1 for 89ZrDFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34±0.47 %ID g-1) of 89ZrDFO-azepin-onartuzumab (n = 4). Conclusion: Experiments demonstrate that photoradiosynthesis is a viable alternative approach for producing 89Zr-radiolabeled antibodies direct in protein formulation buffer which reduces protein aggregation and liver uptake.

Light‐Activated Protein Conjugation and 89Zr‐Radiolabelling with Water‐Soluble Desferrioxamine Derivatives

“…We recently demonstrated that metal ion binding chelates functionalised with a photoactivatable ArN 3 group can be used to prepare radiolabelled antibodies by either a two‐step conjugation and radiolabelling process or by a novel one‐pot approach . Simultaneous 89 Zr‐radiolabelling and light‐induced conjugation using a photoactivatable DFO‐ArN 3 derivative produced viable PET radiotracers (Scheme ) . Additionally, the photochemical conjugation process is compatible with standard formulation buffers, which allows direct synthesis of radiolabelled‐mAbs from approved drugs like Herceptin TM .…”

“…Formulated protein substrates included human serum albumin (HSA), the engineered antibody onartuzumab (formulated as MetMAb TM ), and the humanised IgG 1 immunoglobulin trastuzumab (formulated as Herceptin TM ). Simultaneous (one‐pot) photoradiolabelling reactions were performed in accordance with Scheme …”

Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of 89Zr-radiolabeled monoclonal antibodies

“…In 2019, our group demonstrated that light-induced activation of chelates bearing aryl azide (ArN3) groups and radiolabelled with a variety of different radionuclides, including 68 Ga 3+ and 89 Zr 4+ ions, can produce viable PET radiotracers direct from formulated antibody solutions. [13][14][15][16][17][18][19][20] The underlying concept of bimolecular photochemically induced protein modification is shown in Figure 1. 19,21 The key motivation for developing an alternative protein conjugation and radiolabelling strategy was to eliminate steps that involved pre-purification of the protein and isolation of a functionalised (non-radiolabelled) intermediate molecule.…”

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

“…[32] Our group has also explored the use of photoactivat-able chelates bearinga na ryl azide to make viable 68 Ga and 89 Zr PET radiotracersvia direct functionalisation of mAbs. [33][34][35][36] Here, we demonstrate that the photophysical properties of tetrazoles can be tuned through syntheticcontrol over the arylene substituents to facilitate the rapid and direct, bimolecular protein labellinga tw avelengths in the UVAr egion of the spectrum. Two-step photochemical conjugation and radiolabelling produced 89 Zr-labelled trastuzumab for PET imaging of human epidermal growth-factor receptor 2( HER2/neu)e xpression in tumours.…”

“…used the photo‐induced cycloaddition process and labelled an alkene‐functionalised, tumour‐specific peptide with a modified tetrazole which allowed 68 Ga and 64 Cu radiolabelling for PET and optical imaging in a glioblastoma model [32] . Our group has also explored the use of photoactivatable chelates bearing an aryl azide to make viable 68 Ga and 89 Zr PET radiotracers via direct functionalisation of mAbs [33–36] …”