Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection

Area of Science:

• Biotechnology
• Immunology
• Oncology

Background:

• Traditional antibody therapies face challenges including large IgG size, difficulties in scFv construction, and murine antibody immunogenicity.
• Human scFv selection from phage libraries offers an alternative but often yields low affinities for therapeutic use.
• Chain shuffling, successful for haptens, was explored for protein antigens to improve scFv affinity.

Purpose of the Study:

• To enhance the affinity of a non-immune human scFv targeting the c-erbB-2 tumor antigen using chain shuffling.
• To investigate the impact of light-chain and heavy-chain shuffling on scFv affinity.
• To explore selection strategies for isolating high-affinity scFv, including those that dimerize.

Main Methods:

• Chain shuffling (light and heavy) was employed to modify a human scFv targeting c-erbB-2.
• Selection strategies included antigen immobilization on polystyrene and solution-phase selection with biotinylated antigen.
• Screening methods involved BIAcore analysis for koff measurements and assessment of scFv dimerization.

Main Results:

• Light-chain shuffling increased scFv affinity sixfold (Kd = 2.5 x 10(-9) M), and heavy-chain shuffling increased it fivefold (Kd = 3.1 x 10(-9) M).
• Spontaneously dimerizing scFv were isolated when selection was performed on immobilized antigen, showing increased avidity.
• High-affinity monomeric scFv were obtained through solution-phase selection and koff screening.

Conclusions:

• Chain shuffling is an effective method for increasing the affinity of human scFv against protein antigens like c-erbB-2.
• Selection strategies significantly influence the outcome, with solution-phase selection yielding higher-affinity monomeric scFv.
• This approach enables the production of therapeutic-grade human scFv without the need for immunization, comparable to hybridoma-derived antibodies.