Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting

Conjugates of small molecule and antibodies are broadly employed diagnostic and therapeutic agents. Appending a small molecule to an antibody often significantly impacts the properties of the resulting conjugate. Here we detail a systematic study investigating the effect of various functional groups on the properties of antibody-fluorophore conjugates. This was done through the preparation and analysis of a series of masked heptamethine cyanines (CyMasks) bearing amides with varied functional groups. These were designed to exhibit a broad range of physical properties, and include hydrophobic (-NMe2), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH2)2NMe3+) anionic (NH-(CH2)2SO3-), and zwitterionic (N-(CH2)2NMe3+)-(CH2)3SO3-) variants. The CyMask series was appended to tumor targeting monoclonal antibodies (mAbs) and analyzed for effects on tumor targeting, clearance and non-specific organ uptake. Among the series, zwitterionic and cationic dye conjugates showed the highest tumor-to-background ratio (TBR), although the latter also exhibited an elevated liver-to-background ratio (LBR). Overall, these studies provide a strategy to test the functional group effects and suggest that zwitterionic substituents are an attractive strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.