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

Abstract

Conjugates of small molecules 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 (−NMe 2 ), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH 2 ) 2 NMe 3 + ), anionic (NH-(CH 2 ) 2 SO 3 − ), and zwitterionic (N-(CH 2 ) 2 NMe 3 + )-(CH 2 ) 3 SO 3 − ) variants. The CyMask series was appended to monoclonal antibodies (mAbs) and analyzed for the effects on tumor targeting, clearance, and non-specific organ uptake. Among the series, zwitterionic and pegylated dye conjugates had the highest tumor-to-background ratio (TBR) and a low liver-to-background ratio. By contrast, the cationic and zwitterionic probes had high tumor signal and high 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 possess an optimal combination of high tumor signal, TBR, and low LBR. These results suggest an appealing strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.

Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging

“…Therefore, we sought to introduce additional structural modifications. Our studies and others have found that careful balance and placement of charged functional groups significantly improve the properties of various targeted imaging agents. ,− In particular, our recent studies identified a first-generation FNIR-Tag, which contains an O -alkyl quaternary amine at the C4′ position and improves the tumor targeting of mAb and peptide conjugates. , Here, we explore placing additional charged functional groups adjacent to the polymethine chromophore. With this goal in mind, we designed FNIR-Tag-766 (Figure A), which contains both trimethyl-propyl ammonium indolenine substituents and a N -trimethyl-propyl-ammonium- N -butyl-sulfonate tertiary amide, which we used previously, at the 2-position of the C4′ aryl ring …”

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging

“…In contrast, asymmetric cyanines exhibit a larger Stokes shift with increased fluorescence intensity and brightness because of the unequal electron distribution, resulting in an altered excited state structure. 26–28 Analogous cyanine structures have already been used in haematology. 29…”

“…38 In contrast, asymmetric cyanines exhibit a larger Stokes shift with increased fluorescence intensity and brightness because of the unequal electron distribution, resulting in an altered excited state structure. [26][27][28] Analogous cyanine structures have already been used in haematology. 29 In spite of the fact that the relevance of cyanine conjugated antibodies is obviously high, recently, only a few examples have been published (Table S1 †).…”

Unraveling the Chemistry of meso-Cl Tricarbocyanine Dyes in Conjugation Reactions for the Creation of Peptide Bonds

“…Other authors have previously noted the influence of structure and charge on the pharmacokinetics of cyanine dyes and cyanine−antibody conjugates. 42,43 Further cellular imaging micrographs are given in the Supporting Information (Figures S44−S50). Also, the excitation−emission maps (EEMs) of the merocyanine 2 and the product of the MHI-148 oxidation, MHI-148-O, showed contrasting emission−excitation profiles when measured in PBS or 10% serum medium, as shown in Figure 5.…”

“…The oxo form of the ubiquitous MHI-148 (the nonsulfonated analogous of compound 2 , MHI-148-O ) appears to be readily taken up by cancer cells even at 10 μM concentrations. Other authors have previously noted the influence of structure and charge on the pharmacokinetics of cyanine dyes and cyanine–antibody conjugates. , Further cellular imaging micrographs are given in the Supporting Information (Figures S44–S50).…”