Intracellular and cell surface displayed single-chain diabodies

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Intracellular antibody fragments offer therapeutic potential by inhibiting protein function or trafficking.
• Bivalent and bispecific antibody fragments can enhance inhibitory potential and enable intracellular molecule retargeting.

Purpose of the Study:

• To evaluate the functional expression of intracellular bispecific diabodies.
• To assess the subcellular localization and functional assembly of modified bispecific single-chain diabodies.

Main Methods:

• A bispecific single-chain diabody was engineered for targeting to specific subcellular compartments (cell membrane, ER, mitochondria, cytoplasm, nucleus).
• Subcellular localization was determined using immunofluorescence.
• Functional antibody assembly was assessed by measuring beta-galactosidase binding and substrate conversion.

Main Results:

• Bispecific single-chain diabodies were successfully directed to all targeted subcellular compartments.
• Functional antibody assembly and antigen binding activity were observed only for diabodies localized to the endoplasmic reticulum or cell membrane.
• No functional antigen binding was detected for diabodies in the cytoplasm, nucleus, or mitochondria.

Conclusions:

• Bispecific recombinant antibody fragments can be functionally expressed within the secretory pathway.
• Integration into the plasma membrane allows for functional bispecific antibody activity in mammalian cells.
• Subcellular compartment targeting is critical for the functional expression of intracellular antibody fragments.