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. 2008 Dec;19(12):2527-34.
doi: 10.1021/bc800113v.

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

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Free PMC article

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

Shannon J Sirk et al. Bioconjug Chem. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Schematic representation of CysDb. (A) Gene assembly for anti-CD20 and anti-HER2 CysDb (scFv monomer) with 5-aa linker and cysteine modification shown. (B) CysDb with antigen binding sites indicated by starbursts. CysDb is shown in covalent dimer form, with disulfide bond in the predicted location. (C) Conjugation reaction linking cys-diabody to fluorescent probe.
Figure 2
Figure 2
SDS-PAGE of three CysDbs under non-reducing (left) and reducing (right) conditions. Previously characterized anti-CEA CysDb shown for comparison purposes. Non-reduced samples migrate at expected size for covalent dimer (50 kDa) and reduced samples migrate at expected size for monomeric scFv (25 kDa).
Figure 3
Figure 3
Size exclusion chromatography of cys-diabodies on Superdex 75 column. Previously characterized anti-CEA CysDb shown for comparison purposes.
Figure 4
Figure 4
Flow cytometry of single-channel labeling of Daudi cells. Unlabeled cells (UL) shown in solid grey. (A) Three channels of dye-conjugated anti-CD20 CysDb (Conj., thick line) and mock conjugated dye (Mock, dotted line) demonstrating minimal background from non-specific binding of free dye molecules. (B) Three channels of low (0.5μl, dotted line) and high (5μl, thick line) concentrations of dye-conjugated anti-CD20 CysDb demonstrating concentration-dependent shift in mean fluorescence with increasing quantities of conjugated CysDb. (C) Three channels of dye-conjugated anti-CD20 CysDb (Conj., thick line) with minimal binding to CD20-negative MCF7-HER2 cells, demonstrating antigen-specificity of binding.
Figure 5
Figure 5
Flow cytometry of single-channel labeling of MCF7-HER2+ cells. Unlabeled cells (UL) shown in solid grey. (A) Three colors of dye-conjugated anti-HER2 CysDb (Conj., thick line) and mock conjugated dye (Mock, dotted line) demonstrating minimal background from non-specific binding of free dye molecules. (B) Three channels of low (0.5μl, dotted line) and high (5μl, thick line) concentrations of dye-conjugated anti-HER2 CysDb demonstrating concentration-dependent shift in mean fluorescence with increasing quantities of conjugated CysDb. (C) Three channels of dye-conjugated anti-HER2 CysDb (Conj., solid line) with minimal binding to HER2-negative Daudi cells, demonstrating antigen-specificity of binding.
Figure 6
Figure 6
Flow cytometry of 3-channel labeling of Daudi (A and B) and MCF7-HER2 (C and D) cells with antigen-specific and isotype control dye-conjugated CysDbs. (A) Two representative plots from 3-channel experiments demonstrating detection in FL2 of specific binding of PE-conjugated anti-CD20 CysDb with minimal signal in FL1 or FL4 from co-incubated APC- or A488-conjugated anti-HER2 CysDbs, respectively. (B) Bar graph presenting compiled results of all 3-channel labeling experiments on Daudi cells, demonstrating consistent, specific detection of all three dye-conjugated anti-CD20 CysDbs and minimal background from non-specific binding of dye-conjugated anti-HER2 CysDbs. Analogous experiments were performed with dye-conjugated anti-HER2 CysDbs on MCF7-HER2 cells, using dye-conjugated anti-CD20 CysDbs as isotype controls (C, D).

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