Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation

Area of Science:

• Protein Engineering
• Immunology
• Biotechnology

Background:

• Antibody fragments like Fv are crucial for targeted therapies but often lack stability.
• Current stabilization methods, such as linkers or disulfide bonds, have limitations.
• Developing robust and stable Fv fragments is essential for improving therapeutic efficacy.

Purpose of the Study:

• To develop and characterize a novel method for stabilizing recombinant antibody Fv fragments.
• To create a covalently interconnected Fv fragment, termed permutated Fv (pFv), via base-loop connections.
• To evaluate the antigen-binding properties and stability of the novel pFv fragment.

Main Methods:

• Engineered a permutated Fv (pFv) fragment by covalently linking the V(H) and V(L) domains at their base-loops.
• Constructed a pFv-immunotoxin by fusing the anti-Tac pFv to a truncated Pseudomonas exotoxin.
• Utilized refolding of recombinant inclusion body protein and a renaturation protocol for fragment production.

Main Results:

• The anti-Tac pFv fragment demonstrated retained antigen binding affinity and full specificity compared to anti-Tac scFv.
• The pFv-immunotoxin showed specific cytotoxicity towards antigen-expressing cancer cells.
• The pFv fragment exhibited relative stability, retaining 25% binding activity after 24 hours in human serum at 37°C.

Conclusions:

• Covalent interconnection of Fv domains at base-loops is a viable strategy for stabilizing antibody fragments.
• The developed pFv technology offers a promising alternative to conventional linker or disulfide stabilization methods.
• This novel stabilization approach holds potential for enhancing the utility of Fv fragments in therapeutic applications.