Side by side comparison of NOTA and DOTA for conjugation efficiency, gallium-68 labeling, and in vivo biodistribution of anti-mesothelin sdAb A1-His

Background

Mesothelin is a glycoprotein overexpressed in various cancers, with limited expression in healthy tissues. The single-domain antibody (sdAb, or nanobody) A1-His has previously successfully been validated in mice for the SPECT imaging of mesothelin positive tumors following radiolabeling with ^99mTc. Our objective was to radiolabel this sdAb with ⁶⁸Ga for PET imaging, exhibiting superior sensitivity and resolution than SPECT in clinical practice. To this aim, it was conjugated to NOTA chelator that is commonly employed for ⁶⁸Ga labeling of antibody-derived tracers. In addition, the high affinity and specificity of A1-His sdAb position it as a promising candidate for theranostic applications. In anticipation of future radiolabeling with beta-emitting radionuclides, DOTA-conjugated A1-His was also evaluated. Given the high thermal stability of sdAbs, this DOTA-conjugated sdAb could potentially be implemented in future studies as a theranostic agent with beta-emitting radionuclides.

Results

A1-His was successfully conjugated to p-SCN-Bn-DOTA and p-SCN-Bn-NOTA under optimized conditions, achieving chelator-to-sdAb ratios of 1.8 and 1.3, respectively. NOTA-A1-His allowed rapid radiolabeling with ⁶⁸Ga at room temperature, achieving high radiochemical purity (> 98%) within 5 min. Using DOTA, similar purity was obtained at 60 °C for 15 min. Both radiotracers demonstrated stability over 4 h in the radiolabeling medium and 2 h in human blood. However, some instability was observed in murine blood. Biodistribution and imaging studies in mice bearing mesothelin-expressing tumors showed specific tumor targeting for both tracers. Notably, [68Ga]Ga-DOTA-A1-His exhibited twofold lower kidney uptake compared to [68Ga]Ga-NOTA-A1-His, potentially enhancing imaging contrast and reducing renal radiation exposure. His-tag removal, further improves the biodistribution profile of the 2 tracers.

Conclusions

Both p-SCN-Bn-DOTA and p-SCN-Bn-NOTA chelators can be effectively conjugated to the A1 sdAb and radiolabeled with ⁶⁸Ga, producing stable radiotracers with specific tumor-targeting capabilities. NOTA chelator offers advantages in rapid, room-temperature radiolabeling. However, DOTA would offer the advantage to be employed for theranostic approaches using β⁻ emitters such as ¹⁷⁷Lu or ¹⁶¹Tb. The lower kidney retention of DOTA-A1 also suggests that its dosimetry, a key factor in theranostic, would be more favorable.