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

Abstract

Protein‐conjugates are vital tools in biomedical research, drug discovery and imaging science. For example, functionalised monoclonal antibodies (mAbs) coupled to the desferrioxamine B (DFO) chelate and radiolabelled with 89Zr4+ ions are used as radiopharmaceuticals for diagnostic positron emission tomography (PET). In this context, protein functionalisation requires the formation of a covalent bond that must be achieved without compromising the biological properties of the mAb. Photochemistry offers new synthetic routes toward protein conjugates like 89Zr‐mAbs but to harness the potential of light‐induced conjugation reactions new photoactivatable reagents are required. Herein, we introduce two photoactivatable DFO‐derivatives functionalised with an aryl azide (ArN3) for use in light‐activated conjugation and radiosynthesis of 89Zr‐mAbs. Incorporation of a tris‐polyethylene glycol (PEG)3 linker between DFO and the ArN3 group furnished water‐soluble chelates that were used in the one‐pot, photoradiosynthesis of different 89Zr‐radiolabelled protein conjugates with radiochemical yields up to 72.9±1.9 %. Notably, the DFO‐PEG3 chelates can be readily synthesised in accordance with Good Laboratory Practice (GLP), which will facilitate clinical trials with photoradiolabelled 89Zr‐mAbs.

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

“…The synthesis of radiolabelled mAbs (and other protein-drug conjugates) requires chemical modification of the glycoprotein. 1 Standard protein coupling chemistries rely almost exclusively on thermochemically induced reactions such as the post-translational modification of amino-acid side chains or the site-specific labelling of glycans by chemical or enzymatic methods. [2][3][4][5][6][7][8][9][10] Although highly successful, these classic approaches suffer from a number of important limitations.…”

Microfluidic Preparation of 89Zr-Radiolabelled Proteins by Flow Photochemistry

“…The functionalised DFO-PEG 3 -Et-ArN 3 metal binding chelate (compound 1 ; Scheme 1 ) was synthesised with an overall yield of 24% [ 22 ]. Briefly, treatment of 3-(4-aminophenyl)propanoic acid with imidazole-1-sulphonyl azide HCl [ 23 ], potassium carbonate and a catalytic amount of copper(II) sulphate pentahydrate furnished the corresponding para -substituted aryl azide compound 2 with a 76% yield.…”

“…For comparison, manual reactions were also performed by using direct, top-down irradiation a stirred reaction mixture in a ≈1 mL glass vial. In this manual approach previously reported by Guillou et al, [ 22 ] [ 89 Zr]ZrDFO-PEG 3 -Et-azepin-HSA was produced with a decay-corrected RCY of 59.6 ± 3.6% ( n = 3) and with a RCP > 90%. Further refinement of the microfluidic apparatus and optimisation of the chemical methods is required before the process is competitive with manual reactions.…”

“…We recently reported the photoradiosynthesis of several viable 68 Ga 3+ and 89 Zr 4+ protein-conjugate PET radiotracers, from photoactivatable metal-binding chelates functionalised with an ArN 3 group [ 19 , 20 , 21 , 22 ]. Importantly, this photochemical conjugation process occurs at wavelengths that do not disrupt protein structure or function.…”

“…To enhance the water-solubility of the photoactivatable DFO derivatives, we recently introduced two new compounds (including DFO-PEG 3 -Et-ArN 3 1 ; Scheme 1 ) that link the metal binding chelate to the ArN 3 group via a polar tris -polyethylene glycol (PEG 3 ) linker [ 22 ]. Compound 1 is an excellent ligand for exploring the potential for automated radiosynthesis of 89 Zr-radiolabelled protein conjugates via photochemistry in flow ( Figure 1 ).…”