Shelf-Life of ɛ-Lysyl-3-(Trimethylstannyl)Benzamide Immunoconjugates, Precursors for ²¹¹At Labeling of Antibodies

Abstract

Astatine-211 is possibly the most promising radionuclide for targeted a-particle therapy when it comes to the treatment of occult disseminated cancer. Preclinical research has proven effective, and patient studies have been initiated based on these results. However, a lack of production capacity and the complex radiochemistry of 211 At are major obstacles for research and prospective clinical applications. In the present study, astatination of immunoconjugates, already prepared well in advance before radiolabeling, was performed to investigate the possibility of formulating a kit-like reagent for the production of 211 At radiopharmaceuticals. The shelf-life of e-lysyl-3-(trimethylstannyl)benzamide immunoconjugates was evaluated, that is, the effect of different storage times on the quality of the immunoconjugates. The quality being referred to is the capacity to maintain a good radiochemical yield and good cell-binding property after labeling with 211 At. The stability of the conjugates was found to be pH dependent with high stability at pH ‡ 7 and less stability at pH £ 5.5. The immunoconjugates (based on trastuzumab) could be kept for more than 3 months in a phosphate buffered saline solution (pH 7.4) at 4°C before labeling, without compromising the quality of the labeled product. The conjugates are also unaffected by storage at -20°C. Conjugates with a good shelf-life compatible with distant shipping as well as improved radiochemistry are important steps to facilitate further clinical progress with 211 At.

Synthesis and Evaluation of Astatinated N-[2-(Maleimido)ethyl]-3-(trimethylstannyl)benzamide Immunoconjugates

“…The produced MSB-immunoconjugate in PBS (pH 7.4) was successfully stored for several weeks prior to radiolabeling without any decomposition into fragments, with maintained structural integrity (shown by FPLC and native-PAGE) and without a decrease in labeling performance. The stability of the MSB immunoconjugates was expected according to previous results obtained for ATE-like reagents . High radiochemical yields and high specific activities (see Table ) were obtained using MSB immunoconjugates after refrigerated storage between 12 and 44 days (in air at +4 °C).…”

“…Of the 32 sulfhydryl groups, 8 originate from interchain disulfide bridges, which are exposed to solvent and hence the most easily available for modification. These sulfide groups are thus selectively targeted when performing conjugation with maleimide derivatives after DTT reduction. , The measured number of MSB linkers per antibody was similar to the number of ATE linkers per antibody (6.7 ± 2.4) achieved with the direct ATE conjugation protocol …”

“…12,9 The measured number of MSB linkers per antibody was similar to the number of ATE linkers per antibody (6.7 ± 2.4) achieved with the direct ATE conjugation protocol. 21 The MSB-immunoconjugates of trastuzumab were labeled with astatine-211 (5−30 MBq), according to Figure 2. An average recovery of 77.2 ± 3.1% (no decay correction) was obtained after size exclusion chromatography purification.…”

“…The stability of the MSB immunoconjugates was expected according to previous results obtained for ATE-like reagents. 21 High radiochemical yields and high specific activities (see Table 1) were obtained using MSB immunoconjugates after refrigerated storage between 12 and 44 days (in air at +4 °C). In addition, the MSB immunoconjugate could be lyophilized and redissolved in buffer prior to successful astatine labeling without any decrease in immunoreactivity (expressed as the immunoreactive fraction, IRF) as compared to a reference immunoconjugate stored in the refrigerator (IRF (Lyophilized) = 0.93; IRF (Reference) = 0.86).…”

The Different Strategies for the Radiolabeling of [211At]-Astatinated Radiopharmaceuticals

“…In recent years, the primary approach for radiolabelling proteins with 211 At involves a two-step procedure based on the radiosynthesis of an intermediate prosthetic group N-[ 211 At]succinimidylastatobenzoate ([ 211 At]SAB), by the electrophilic astatodestannylation of the corresponding aryltrialkylstannane precursor, similar to the analogous procedure that had been developed previously for radioiodination [71]. Emma Aneheim et al [72] indicated that immunoconjugates formed by conjugating an antibody (e.g., Traztuzumab) with alkyltin-based prosthetic groups (N-succinimidyl 3-(trimethylstannyl)benzoate) can be stored in a neutral buffer (pH 7.4) for over 3 months at 4 • C and they can be stored at −20 • C without compromising the quality of the labeled product. Lindegren et al [73,74] developed a single-step strategy from the precursor grafted to the antibody prior, thereby saving time and enhancing yields compared to two-step approaches.…”