Development of a novel antibody–tetrazine conjugate for bioorthogonal pretargeting

Abstract

Recently, bioorthogonal chemistry based on the Inverse Electron-Demand Diels-Alder (IEDDA) cycloaddition between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) analogues added an interesting dimension to molecular imaging. Until now, antibodies (Abs) were tagged with TCO and after pretargeting they were reacted with tetrazines substituted with reporters. However, TCO tags have the tendency to degrade under physiological conditions, and due to their hydrophobic nature are buried within the protein. This results in loss of reactivity and a low Ab functional loading. To circumvent these problems, we report for the first time an approach in which tetrazines are used as tags for antibody (Ab) modification, and TCO as the imaging agent. We developed a new Ab-tetrazine conjugate, which displays a high functional loading, good stability and reactivity. We utilized this immunoconjugate for live-cell imaging together with novel TCO probes, resulting in selective and rapid labeling of SKOV-3 cells. Our approach may be useful for in vivo pretargeted imaging.

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

“…and Sobott et al. reported on a method allowing to determine the intact mass of an ADC composed of noncovalently associated heavy and light chains, with a drug linked to interchain cysteine residues . Debaene et al.…”

“…Adapted with permission from [143] F I G U R E 8 Native MS spectra of IgGs (CNTO5825 and NIST) obtained on a Q-TOF-2 instrument (Waters) after direct infusion (A and B) and after SEC analysis, indicating partial unfolding of the antibodies (without breaking disulfide bonds). LC conditions: Flow rate = 0.1 mL/min; mobile phase = 100 mM ammonium acetate, pH 6.8; using a 4.6 mm id × 100 mm BEH SEC column packed with 1.7 μm particles (200Å pores) with a drug linked to interchain cysteine residues [49,155]. Debaene et al conducted native MS experiments of Brentuximab vedotin (also an interchain cysteinyl-linked ADC) providing accurate mass measurements of intact ADCs together with the average DAR and drug distribution [156].…”

Click‐Chemistry‐Mediated Rapid Microbubble Capture for Acute Thrombus Ultrasound Molecular Imaging

“…Inverse‐electron‐demand Diels–Alder cycloaddition between a 1,2,4,5‐tetrazine and the trans ‐cyclooctene (TCO) system is an example of a highly selective and rapid bioorthogonal coupling process that has been successfully used to prepare targeted nuclear and optical molecular imaging probes . Through using a bioorthogonal reaction, this pretargeted molecular imaging strategy has become a powerful tool for in vitro and in vivo imaging . In a typical bioorthogonal pretargeting approach, the target is first bound with a TCO‐modified antibody, and then a tetrazine probe is introduced in order to achieve labeling through cycloaddition.…”

“…[11][12][13] Through using ab ioorthogonal reaction, this pretargeted molecular imaging strategy has become ap owerful tool for in vitro and in vivo imaging. [14] In at ypical bioorthogonal pretargeting approach, the target is first bound with aT CO-modified antibody,a nd then at etrazine probe is introduced in order to achieve labelingt hroughc ycloaddition.Thrombosis is developed through blood vessel wall damage or through ap athological process that inducesa ctivation of coagulation factor to cause accumulation of platelets and fibrin. [15] Although many imaging strategies-including magnetic resonancei maging (MRI), [16] ultrasound, [17] computed tomography (CT), [18] etc.-for visualization of the thrombusf ormationp rocess have been developed, the specific detection and rapid evaluationo ft hrombotic events, especially at the molecular level, still presents am ajor clinicalc hallenge.…”

“…[11][12][13] Through using ab ioorthogonal reaction, this pretargeted molecular imaging strategy has become ap owerful tool for in vitro and in vivo imaging. [14] In at ypical bioorthogonal pretargeting approach, the target is first bound with aT CO-modified antibody,a nd then at etrazine probe is introduced in order to achieve labelingt hroughc ycloaddition.…”