Site-selective radiolabeling using mushroom tyrosinase and the strain-promoted oxidation-controlled 1,2-quinone cycloaddition

Abstract

We report the in vitro characterization and in vivo evaluation of a novel ⁸⁹Zr-labeled radioimmunoconjugate synthesized using a site-selective bioconjugation strategy based on the oxidation of tyrosinase residues exposed by the deglycosylation of the IgG and the subsequent strain-promoted oxidation-controlled 1,2-quinone cycloaddition between these amino acids and trans-cyclooctene-bearing cargoes. More specifically, we site-selectively modified a variant of the A33 antigen-targeting antibody huA33 with the chelator desferrioxamine (DFO), thereby producing an immunoconjugate (DFO-^SPOCQhuA33) with equivalent antigen binding affinity to its parent immunoglobulin but attenuated affinity for the FcγRI receptor. This construct was subsequently radiolabeled with [⁸⁹Zr]Zr⁴⁺ to create a radioimmunoconjugate - [⁸⁹Zr]Zr-DFO-^SPOCQhuA33 - in high yield and specific activity that exhibited excellent in vivo behavior in two murine models of human colorectal carcinoma.

Scheme 1. (A) The oxidation of tyrosine by mushroom tyrosinase; (B) the strain-promoted oxidation-controlled 1,2-quinone cycloaddition.

Scheme 2. Schematic of the deglycosylation of an antibody followed by the mTyr- and SPOCQ cycloaddition-mediated modification of these residues with TCO-DFO.

Fig. 1. Protein-stained and fluorescent SDS-PAGE gel electrophoresis of huA33 in various reaction conditions.

Fig. 2. ELISA assays exploring the binding of huA33, ^PNGaseFhuA33, DFO-huA33, and DFO-^SPOCQhuA33 with (A) the A33 antigen and (B) FcγRI as well as (C) a cell-based immunoreactivity assay of [⁸⁹Zr]Zr-DFO-huA33 and [⁸⁹Zr]Zr-DFO-^SPOCQhuA33 with A33 antigen-expressing SW1222 colorectal cancer cells (n = 3).

Fig. 3. In vivo evaluation of the radioimmunoconjugates. Representative maximum intensity projection PET scans collected 24, 72, and 120 h after the intravenous administration of [⁸⁹Zr]Zr-DFO-huA33 or [⁸⁹Zr]Zr-DFO-^SPOCQhuA33 [3.7–4.0 MBq (100–110 μCi), 20–22 μg, in 100 μL of PBS] to athymic nude or NSG mice bearing subcutaneous A33-expressing SW1222 colorectal cancer xenografts (n = 4).