Abstract
Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.
Highlights
There are a number of approaches to chemical modification of antibodies, especially with fluorescent dyes [1,2,3,4,5,6] and therapeutic agents [7,8,9,10,11,12]
We aimed to develop a convenient procedure for labeling any immunoglobulin G via periodate oxidation followed by oxime ligation
Compound 2 was acylated by oxysuccinimide esters of fluorescent dyes—BODIPY FL (BDP-FL), 6-carboxyfluorescein (FAM), and Alexa 488 (AF488)—to yield protected derivatives 3–5 (Scheme 1)
Summary
There are a number of approaches to chemical modification of antibodies, especially with fluorescent dyes [1,2,3,4,5,6] and therapeutic agents [7,8,9,10,11,12]. Carbonyl groups suitable for oxime ligation can be introduced into the antibody molecule by periodate oxidation of the glycosylated site [5,23,24], even one that has been genetically engineered [25]. The carbonyl group can be introduced into the antibody by enzymatic modification of the glycan part with a keto-sugar [26], or via site-specific incorporation of genetically encoded p-acetylphenylalanine into a peptide chain [27,28]. Whereas the two latter approaches require cumbersome enzymatic or genetic manipulations, periodate oxidation of the carbohydrate site can be applied to any full-size antibody. The glycosylation site of immunoglobulin G is located at the heavy chain, at a significant distance from the antigen binding domain, the modification does not influence antibody affinity
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