Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

Abstract

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal 89Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with 89Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing 89Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to high selective tumor uptake (67.5 ± 5.0 %ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer

“…The 5B1 was modified to incorporate four azido groups ( ss 5B1-N 3 ) by a previously reported method (25,26) and then was incubated with DIBO-DFO, DIBO-FL, or a 1:1 mixture of the two constructs to create the completed immunoconjugates: ss DFO-5B1, ss FL-5B1, and ss Dual-5B1. An SDS/PAGE assay was performed, as previously described (25,26), to demonstrate the specificity of the conjugation. For biodistribution and imaging studies, the Zrss dual-5B1 (13,28,29).…”

“…Those shortcomings include the loss of immunoreactivity due to conjugation at the antigen-binding region, random conjugation that leads to poorly defined constructs, and an intrinsic lack of reproducibility, as well as laborious, costly optimization of each novel construct. The combination of glycan engineering and bioorthogonal "click" chemistry has proved a successful strategy for conjugating molecules distal to the antigenbinding region of mAbs in a manner that is highly specific and reproducible, circumventing the aforementioned problems (25,26). Specifically, using a site-specific conjugation strategy for affixing chelator and/or dye molecules via the heavy chain glycans leads to well-defined, robust immunoconjugates in a highly reproducible manner that requires minimal optimization and results in a minimal loss of immunoreactivity.…”

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…In a first step, terminal galactose units are removed by β1,4-galactosidase to yield homogeneous G0 species which are then reglycosylated to a uniform G2 species by a mutant β1,4-galactosyltransferase-mediated attachment of C-2 modified galactoses, e.g., 2-keto-galactose (C2-keto-Gal) or 2-azido-galactose (C2-GalNAz). While Boeggeman et al applied this technology to develop biotinylated and fluorescently-labeled antibody conjugates [104,105], other groups employed the protocol to generate radioactive antibodies as in vivo imaging agents [106,107] or ADCs [108]. Van Geel and co-workers took this technology one step further by removing the terminal galactose residues and trimming the entire carbohydrate moiety down to its core GlcNAc unit.…”

Conjugados de ovalbumina e albumina bovina com desferrioxamina e suas interações com íons metálicos

“…Esses conjugados podem potencialmente desempenhar papeis como fármacos, especialmente em radioterapia, além da desmineralização da ferritina. Complexos de proteínas ligadas à desferrioxamina apresentam estabilidade in vivo [26][27][28][29].…”

“…Esses conjugados podem potencialmente desempenhar papeis como fármacos, especialmente em radioterapia, além da desmineralização da ferritina. Complexos de proteínas ligadas à desferrioxamina apresentam estabilidade in vivo [26][27][28][29].Dentre as vantagens de se utilizar tais moléculas é que a desferrioxamina conjugada pode proteger as células do stress oxidativo, uma vez que impede a reação de Fenton [30].Além da reação esperada com ferro, ainda pode haver reações com metais como zircônio, gálio e gadolínio [31][32][33].Conjugados com desferrioxamina modificada quimicamente com glutaraldeído, utilizando albumina humana como biopolímero, apresentam alta afinidade a gálio [34].A ovoalbumina (OVA) e a albumina sérica bovina (BSA) são solúveis em água e com capacidade natural de ligação com ferro, mas com muito baixa afinidade [35]. A ovoalbumina está presente na clara do ovo de galinha em uma proporção que pode variar entre 60-65%.…”