The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre‐association (non‐covalent ligand‐protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.

A method for the simultaneous (one-step) photochemical conjugation and 89 Zr-radiolabeling of antibodies is introduced. A photoactivatable chelate based on the functionalization of desferrioxamine B with an arylazide moiety (DFO-ArN 3 , [1]) was synthesized. The radiolabeled complex, 89 Zr-1 + , was produced and characterized. Density functional theory calculations were used to investigate the mechanism of arylazide photoactivation. 89 Zr-radiolabeling experiments were also used to determine the efficiency of photochemical conjugation. A standard two-step approach gave a measured conjugation efficiency of 3.5% G 0.4%. In contrast, the one-step process gave a higher photoradiolabeling efficiency of $76%. Stability measurements, cellular saturation binding assays, positron emission tomographic imaging, and biodistribution studies in mice bearing SK-OV-3 tumors confirmed the biochemical viability and tumor specificity of photoradiolabeled [ 89 Zr]ZrDFO-azepin-trastuzumab. Experimental data support the conclusion that the combination of photochemistry and radiochemistry is a viable strategy for producing radiolabeled proteins for imaging and therapy.

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.

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Most experimental work in the space of bioconjugation chemistry focuses on using new methods to construct covalent bonds between a cargo molecule and a protein of interest such as a monoclonal antibody (mAb). Bond formation is important for generating new diagnostic tools, yet when these compounds advance to preclinical in vitro and in vivo studies, and later for translation to the clinic, understanding the fate of potential metabolites that arise from chemical or enzymatic degradation of the construct is important to obtain a full picture of the pharmacokinetic performance of a new compound. In the context of designing new bioconjugate methods for labeling antibodies with the positron-emitting radionuclide 89Zr, we previously developed a photochemical process for making 89Zr-mAbs. Experimental studies on [89Zr]­ZrDFO-PEG3-azepin-mAb constructs revealed that incorporation of the tris-polyethylene glycol (PEG3) linker improved the aqueous phase solubility and radiochemical conversion. However, the use of a PEG3 linker also has an impact on the whole-body residence time of the construct, leading to a more rapid excretion of the 89Zr activity when compared with radiotracers that lack the PEG3 chain. In this work, we investigated the metabolic fate of eight possible metabolites that arise from the logical disconnection of [89Zr]­ZrDFO-PEG3-azepin-mAb at bonds which are susceptible to chemical or enzymatic cleavage. Synthesis combined with 89Zr-radiolabeling, small-animal positron emission tomography imaging at multiple time points from 0 to 20 h, and measurements of the effective half-life for whole-body excretion are reported. The conclusions are that the use of a PEG3 linker is non-innocent in terms of its impact on enhancing the metabolism of [89Zr]­ZrDFO-PEG3-azepin-mAbs. In most cases, degradation can produce metabolites that are rapidly eliminated from the body, thereby enhancing image contrast by reducing nonspecific accumulation and retention of 89Zr in background organs such as the liver, spleen, kidney, and bone.