Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

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.

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody–Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

“…Dichloromethane (DCM) and methanol (MeOH) were purified by distillation, dimethyl sulfoxide (DMSO) was purified by vacuum distillation and stored over 3 Å molecular sieves. Compounds 4 [ 35 ], 15 [ 56 ], 19 [ 35 ], 20 [ 57 ], 23 [ 58 ], 26 [ 57 ], 28 [ 31 ], and 29 [ 56 ] were prepared as described previously. Yields refer to chromatographically and spectroscopically ( 1 H and 13 C NMR) homogeneous materials, unless otherwise stated.…”

“…However, the high reactivity of oxyamines even in salt form reduces the panel of available reagents. Recently, we found that the 1-ethoxyethylidene-protecting group on oxyamines allows for performing oxime ligation on periodate-oxidized IgG in situ in an acidic acetate buffer with pH 3.6, with such protected oxyamine derivatives of fluorescent dyes having a virtually unlimited shelf lifetime [ 31 ]. These findings prompted us to apply protected dye oxyamines to the ADC technology.…”

“…Overexpression of the membrane form of PRAME in tumors and its low levels in the cytosol of normal somatic tissues make it a promising target for cancer therapy. In our previous works, we showed the possibility of using fluorescent antibodies to the PRAME protein obtained by oxime ligation as a molecular diagnostic tool [ 31 , 34 , 35 ]. We demonstrated that fluorescent anti-PRAME antibodies allow for immunotyping bone marrow cells of patients with acute leukemia for the presence of the PRAME protein [ 31 ], which significantly affects the course of the disease and further prognosis [ 36 , 37 , 38 , 39 , 40 , 41 , 42 ].…”

Introduction of Carbonyl Groups into Antibodies

“…An alternative approach involves directly conjugating the target molecule with the antibody in one step, eliminating the need for additional procedures (Figure 22). Antibody conjugates with dyes [137,216,232,254,255], cytotoxic drugs such as auristatins [169,221,241,243,256,257] and pyrrolobenzodiazepines [220], folic acid [258], PSMA (prostate-specific membrane antigen) ligands [242], and LXR (liver X receptor) agonists [259] have been generated using this approach. In addition, numerous antibody conjugates with oligonucleotides have been developed, demonstrating the versatility of this method [238][239][240]260] (Figure 23).…”