Click Chemistry Enables [89Zr]Zr-DOTA Radioimmunoconjugation for Theranostic 89Zr-immunoPET

Abstract

There have been predictions that the use of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in zirconium-89 (⁸⁹Zr) immuno-positron emission tomography (⁸⁹Zr-immunoPET) could enhance the in vivo stability of ⁸⁹Zr radioimmunoconjugates. However, conjugating [⁸⁹Zr]Zr-DOTA to a monoclonal antibody (mAb) remains a challenge as the heat treatment required for [⁸⁹Zr]Zr-DOTA chelation can lead to thermal denaturation of the mAb moieties. We developed a method for synthesizing [⁸⁹Zr]Zr-DOTA-mAb based on a tetrazine (Tz)-conjugated bifunctional DOTA derivative 2,2',2″-(10-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,21,26-trioxo-6,9,12,15,18-pentaoxa-29-carboxy-2,22,25-triazanonacosane-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-Tz) and the inverse electron-demand Diels-Alder (IEDDA) click chemistry reaction where trans-cyclooctene-modified mAbs are conjugated to [⁸⁹Zr]Zr-DOTAGA without being exposed to heat. The stability of IEDDA-derived [⁸⁹Zr]Zr-DOTAGA-trastuzumab was confirmed by in vitro, ex vivo, and in vivo testing and comparative analysis against the conventional deferoxamine (DFO) counterpart [⁸⁹Zr]Zr-DFO-trastuzumab. The in vivo immunoPET imaging using [⁸⁹Zr]Zr-DOTAGA-trastuzumab clearly visualized human epidermal growth factor receptor 2-positive malignancies in murine xenograft models. Greater tumor contrast was observed from [⁸⁹Zr]Zr-DOTAGA-trastuzumab at a 72-h delayed scan compared with [⁸⁹Zr]Zr-DFO-trastuzumab. These findings suggest that our IEDDA ligation approach can be an effective means of synthesizing [⁸⁹Zr]Zr-DOTA-mAb and can enhance the theranostic potential of ⁸⁹Zr-immunoPET in DOTA-mediated radioimmunotherapy.

Scheme 1. Synthetic Scheme for the IEDDA Precursors (A) DOTAGA-Tz and (B) TCO-Trastuzumab

DOTAGA-Tz was synthesized through amidation of NH₂-DOTAGA by Tz-PEG₅-NHS ester in dimethylformamide (DMF) with triethylamine (Et₃N). TCO-trastuzumab was prepared by conjugating trastuzumab to TCO-NHS ester.

Figure 1

Deconvoluted zero-charge mass distribution profiles of (A) intact trastuzumab, (B) TCO-trastuzumab, and (C) DFO-trastuzumab. LAR and CAR stand for linker-to-antibody ratio and chelator-to-antibody ratio, respectively.

Scheme 2. Scheme for the Two-Step IEDDA-Driven Synthesis of [⁸⁹Zr]Zr-DOTAGA-Trastuzumab

Figure 2

Spectral intensities of (A) [⁸⁹Zr]Zr-DOTAGA-trastuzumab and (B) [⁸⁹Zr]Zr-DFO-trastuzumab samples purified by size-exclusion chromatography.

Figure 3

Lindmo plots showing the immunoreactivities of (A) [⁸⁹Zr]Zr-DOTAGA-trastuzumab and (B) [⁸⁹Zr]Zr-DFO-trastuzumab against SK-OV-3 cells. The term [cell concentration]⁻¹ denotes the inverse of SK-OV-3 cell concentration, and [cell-binding fraction]⁻¹ the inverse of the fraction of immunocomplexes formed by the radioimmunoconjugates in question and SK-OV-3 cells.

Figure 4

Biodistribution profiles of (A) [⁸⁹Zr]Zr-DOTAGA-trastuzumab and (B) [⁸⁹Zr]Zr-DFO-trastuzumab at 24, 72, and 144 h after administration (P.I.). The uptake data are presented as the mean ± standard deviation with a sample size of n = 4 at each postinjection time point for each radioimmunoconjugate.

Figure 5

PET images of SK-OV-3 xenograft mice at 24, 72, and 144 h after administration with [⁸⁹Zr]Zr-DOTAGA-trastuzumab (upper) and [⁸⁹Zr]Zr-DFO-trastuzumab (lower).