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

Abstract

Efficient methods to functionalize proteins are essential for the development of many diagnostic and therapeutic compounds, such as fluorescent probes for immunohistochemistry, zirconium-89 radiolabeled mAbs (89Zr-mAbs) for positron emission tomography and antibody-drug conjugates (ADCs). This protocol describes a step-by-step procedure for the light-induced functionalization of proteins with compounds bearing the photochemically active aryl azide group. As an illustration of the potential utility of our approach, this protocol focuses on the synthesis of 89Zr-mAbs using photoactivatable derivatives of the metal ion binding chelate desferrioxamine B (DFO). The light-induced synthesis of 89Zr-mAbs is a unique, one-pot process involving simultaneous radiolabeling and protein conjugation. The photoradiochemical synthesis of purified 89Zr-mAbs, starting from unmodified proteins, [89Zr][Zr(C2O4)4]4-(89Zr-oxalate), and a photoactivatable DFO derivative, can be performed in <90 min. The method can be easily adapted to prepare other radiolabeled proteins, ADCs or fluorescently tagged proteins by using drug molecules or fluorophores functionalized with photoactive moieties.

Photoactivatable bis(thiosemicarbazone) derivatives for copper-64 radiotracer synthesis

“…Protein aggregation, which appears as a minor peak or shoulder to shorter retention time in the radio-HPLC chromatograms was <5%, and when compared to the analysis of the protein stock solutions, did not increase after the photoradiosynthesis. Like our previous photoconjugation studies bearing ArN 3 functionalities, 22,41 the addition of DMSO (<1% final volume) does not interfere with the photoactivation or protein-conjugation steps.…”

“…These data confirm that photon absorption is the rate determining step in the photodegradation which is consistent with previous observations. 19,39 Indeed, these two compounds showed higher photo-reactivity than the previously reported photoactive derivatives of aza-macrocyclic, 19,40 acyclic desferrioxamine B (DFO), 20–22,41 N , N ′-bis[2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine- N , N ′-diacetic acid (HBED-CC) 42 and diethylenetriamine pentaacetate (DTPA) 23 chelates bearing ArN 3 groups. Interestingly, photochemical activation of Cu- 7 in water was found to be ∼4.3 times faster than in EtOH, and ∼5.6 times faster than in DMF.…”

“…Expanding on our recent developments in the use of photochemically driven reactions to create functionalised bioconjugates, 19–23 we decided to adapt our photoradiosynthesis methods for use with copper radionuclides. Radiotracers constructed from 64 Cu- or 67 Cu-radiolabelling of monoclonal antibodies (mAb) or related protein fragments could find potential applications for theranostic PET imaging and radioimmunotherapy.…”

“…15 Keinänen et al also reported the sequential pre-targeted 64 Cu PET imaging and 67 Cu-radionuclide therapy in mouse models bearing subcutaneous tumours derived from the SW1222 human colon adenocarcinoma cell line. 18 Expanding on our recent developments in the use of photochemically driven reactions to create functionalised bioconjugates, [19][20][21][22][23] we decided to adapt our photoradiosynthesis methods for use with copper radionuclides. Radiotracers constructed from 64 Cu-or 67 Cu-radiolabelling of monoclonal antibodies (mAb) or related protein fragments could find potential applications for theranostic PET imaging and radioimmunotherapy.…”

Automated Light-Induced Synthesis of 89Zr-Radiolabeled Antibodies for Immuno-Positron Emission Tomography

“…Recently, we developed a one-pot route that combines the bioconjugation and radiolabeling steps using new DFO-chelates bearing a photoactivatable aryl azide group (ArN 3 ) (23)(24)(25)(26). Three key features of this alternative photoradiochemical method for making 89 Zr-mAbs are: i) rapid reaction times that depend on the rate-limiting photoactivation step; ii) high chemical tolerance of the light-induced bioconjugation process with many components of mAb formulation buffers, and iii) avoidance of the need to use prefunctionalized DFO-mAbs.…”