Simultaneous Photoradiochemical Labeling of Antibodies for Immuno-Positron Emission Tomography

Abstract

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.

Photochemical Reactions in the Synthesis of Protein–Drug Conjugates

“…[61] Our laboratory recently reported the synthesis, radiolabelling, and bimolecular couplingo fp hotoactivatable chelates bearingt he photoactive aryl azide (ArN 3 )g roup to antibodies for use in immuno-PET and RIT ( Figure 2). [41][42][43][44][45] Remarkably, switching the conjugation chemistry from traditional, thermally mediated reactions [10][11][12][13] to ap hoto-initiated reaction allowed us to produce radiolabelled mAbs in as imultaneous( one-pot) process directly from fully formulated antibodies (including Figure 1. Timelines howing the evolution of different photoactive groups used in PAL.…”

“…Eur.J. 2020,26,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] www.chemeurj.org Concept the photochemistry for bimolecular protein modification. [67,68] The conformational flexibility of an extended alkyl chain also means that aliphatic carbenes are more likely to undergo intramolecular reactions than the more conformationally rigid aryl carbenes.T hese competing intra-and intermolecularp rocesses are likely to restrict the use of aliphatic diazirines to systems that pre-associate.…”

“…As a proof‐of‐principle, our group recently introduced an alternative approach for the synthesis of radiolabelled antibodies (mAbs) that combined a photochemical activation step with simultaneous radiolabelling to produce viable radiotracers for immuno‐positron emission tomography (immuno‐PET) . Termed, “photoradiosynthesis”, the concept involves light‐induced activation of a photoactive substrate to form a highly reactive electrophile that reacts in situ with nucleophilic amino acid residues (primarily the ϵ‐NH 2 side chain of lysine) on a protein (Scheme ).…”

“… Illustration of the conventional (top) and simultaneous (bottom) route for the conjugation and radiolabelling of biologically active molecules. The concept of “photoradiosynthesis” involves the simultaneous, one‐pot photochemically induced functionalisation and radiolabelling of a biologically active molecule in situ, without the need for pre‐purification …”

“…As ap roof-of-principle, our group recentlyi ntroduced ana lternative approach for the synthesis of radiolabelled antibodies (mAbs) that combined ap hotochemical activation step with simultaneousr adiolabelling to produce viable radiotracersf or immuno-positron emissiont omography( immuno-PET). [41][42][43][44][45] Te rmed, "photoradiosynthesis", the concept involves light-induced activation of ap hotoactive substrate to form ah ighly reactive electrophile that reacts in situ with nucleophilic amino acid residues (primarily the e-NH 2 side chain of lysine) on a protein (Scheme 1). This combination of photochemistry and radiochemistry was inspiredb y, i) the need to performf unctionalisation reactions as quickly as possible when working with radionuclides that have short half-lives, [46] ii)eliminating the isolation and characterisation of afunctionalised intermediate prior to the radiolabelling step, iii)the developmentofaradiolabelling methodt hat is compatible with standard formulation buffers usedt os tabilise mAbs in clinical-grade products, and iv) development of ap rocess that is amenable to full automation.…”