Figure 1
CD19xCD3 DART and BiTE molecules are highly purified and capable of simultaneous engagement of both targets. (A) Schematic representation of DART (left) and BiTE (right) molecules. The DART molecule consists of 2 covalently linked polypeptide chains that heterodimerize to generate 2 specificities. The BiTE molecule is a single polypeptide that assembles to form 2 scFv domains joined by a short linker. (B) DART and BiTE molecules were purified by affinity chromatography followed by SEC. Purified material forms a single peak, as demonstrated by analytical SEC. Elution volume versus absorbance (mAU, milliabsorbance units) is plotted for both molecules. The approximately 15-mL elution volume is consistent with a 50-kDa molecular mass predicted for both DART and BiTE molecules. The black trace is the DART molecule and the gray trace is the BiTE molecule. (C) Nonreducing and reducing SDS-PAGE of purified molecules. Under nonreduced conditions, both DART and BiTE molecules migrate at approximately 50 kDa. A small amount of reduced material can be detected for the DART molecule around 25 kDa. However, these chains are associated in a dimeric complex, as indicated by the SEC profile. Under reducing conditions, the individual chains of the DART molecule migrate at approximately 25 kDa, whereas the single chain of the BiTE molecule remains at approximately 50 kDa. (D) Bispecific ELISA demonstrates simultaneous engagement of both target antigens by the molecules. ELISA plates were coated with soluble human CD3ϵ/δ, then the DART or BiTE molecule was applied at the indicated concentrations. Finally, soluble human CD19-Fc fusion protein was bound, detected with peroxidase-conjugated goat anti–human Fc polyclonal antibody, and developed using a chemiluminescent HRP substrate. The black trace is the DART molecule and the gray trace is the BiTE molecule.

CD19xCD3 DART and BiTE molecules are highly purified and capable of simultaneous engagement of both targets. (A) Schematic representation of DART (left) and BiTE (right) molecules. The DART molecule consists of 2 covalently linked polypeptide chains that heterodimerize to generate 2 specificities. The BiTE molecule is a single polypeptide that assembles to form 2 scFv domains joined by a short linker. (B) DART and BiTE molecules were purified by affinity chromatography followed by SEC. Purified material forms a single peak, as demonstrated by analytical SEC. Elution volume versus absorbance (mAU, milliabsorbance units) is plotted for both molecules. The approximately 15-mL elution volume is consistent with a 50-kDa molecular mass predicted for both DART and BiTE molecules. The black trace is the DART molecule and the gray trace is the BiTE molecule. (C) Nonreducing and reducing SDS-PAGE of purified molecules. Under nonreduced conditions, both DART and BiTE molecules migrate at approximately 50 kDa. A small amount of reduced material can be detected for the DART molecule around 25 kDa. However, these chains are associated in a dimeric complex, as indicated by the SEC profile. Under reducing conditions, the individual chains of the DART molecule migrate at approximately 25 kDa, whereas the single chain of the BiTE molecule remains at approximately 50 kDa. (D) Bispecific ELISA demonstrates simultaneous engagement of both target antigens by the molecules. ELISA plates were coated with soluble human CD3ϵ/δ, then the DART or BiTE molecule was applied at the indicated concentrations. Finally, soluble human CD19-Fc fusion protein was bound, detected with peroxidase-conjugated goat anti–human Fc polyclonal antibody, and developed using a chemiluminescent HRP substrate. The black trace is the DART molecule and the gray trace is the BiTE molecule.

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