Site-Specific, Thiol-Mediated Conjugation of Fluorescent Probes to Cysteine-Modified Diabodies Targeting CD20 or HER2

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Abstract
Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the utility of a C-terminal cysteine modification for site-specific radiolabeling of an anti-CEA diabody, resulting in the creation of a cys-diabody (CysDb). In the present work, the adaptability of the CysDb system was explored by creating two additional CysDbs: one specific for CD20 and one for HER2. Purified CysDbs of both specificities demonstrated behavior consistent with stable, covalent dimers harboring a readily reducible disulfide bond. Each CysDb was site-specifically conjugated to three different fluorophores for optical detection: the large fluorescent proteins phycoerythrin (PE) and allophycocyanin (APC), and the small fluorescent molecule Alexa Fluor488. Fluorophore-conjugated CysDbs bound specifically to their targets in both antigen systems and with each different fluorescent tag as determined by flow cytometry. In vitro specific antigen binding was observed in the presence of a mixture of specific and nonspecifically conjugated CysDbs. Conjugates retained both specificity and fluorescence, demonstrating the successful expansion of the CysDb repertoire to new targets and to new site-specific conjugation possibilities.

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