Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations

Abstract

In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a 99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate—polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig’s immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). 99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. 99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of 99mTc-anti-CD20 IgG-SH in CD20+ tumor versus CD20- tumor. Radiolabeling of derivatized IgG with 99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is “antibody safe” and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.

Radiolabelled polymeric IgA: from biodistribution to a new molecular imaging tool in colorectal cancer lung metastases

“…To develop a soft 99m Tc radiolabelling method applicable to IgA, which preserves the biological functions of antibody and is suitable for low-concentration samples of mAbs, we selected the method initially developed by Biechlin et al [ 22 ] and subsequently optimised and evaluated by Carpenet et al [ 20 ]. This method involved two steps: Ig derivatisation with a synthon, 2-IT, and Ig-SH radiolabelling by an indirect method using the 99m Tc-tricarbonyl core.…”

“…The radiolabelling method used has been described previously [ 20 ]. Briefly, the first step was thiol-derivatisation of Ig with 2-IT.…”

“…This short half-life is not optimal for IgG (serum half-life 20 days) radiolabelling but seems suitable considering the shorter half-life of IgA (3–6 days). In a previous study, we showed that indirect radiolabelling using a bifunctional chelating agent, such as the tricarbonyl core [Tc(CO) 3 (H 2 O) 3 ] + , and a spacer such as 2-iminothiolane (2-IT), provides good radiolabelling yields with high specific activity, and preserves IgG structure and immunoaffinity with limited amounts of antibody [ 20 ].…”

Radiolabeling and Quantitative In Vivo SPECT/CT Imaging Study of Liposomes Using the Novel Iminothiolane-99mTc-Tricarbonyl Complex

“…The prepared liposome sample was derivatized through the primary amine group of DOPE. The free amino group of DOPE was converted into mercaptobutyrimidyl group using 2-iminothiolane (2-IT or Traut's reagent, Sigma-Aldrich, Hungary) [ 29 – 31 ]. 900 µ L of liposome suspension containing 2.42 μ mol of DOPE was mixed with 100 µ L stock solution of 2-IT (14 mM solution in PBS) corresponding to 1.4 µ mol of 2-IT.…”

“…In the present study, a model liposome system composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids is chosen to demonstrate the efficiency of the labeling with the novel iminothiolane- 99m Tc-tricarbonyl complex [ 29 – 31 ]. The studied liposome sample mimics the lipid composition of two clinically approved liposomal products, namely, the Epaxal® and Inflexal V® [ 32 – 35 ].…”