Human anti-self antibodies with high specificity from phage display libraries

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Human antibody fragments binding foreign antigens can be isolated from peripheral blood lymphocytes (PBLs).
• V-genes from PBL mRNA can be shuffled and displayed as single-chain Fv (scFv) fragments on phage for antigen selection.

Purpose of the Study:

• To investigate the isolation of scFv fragments binding human self-antigens from a phage library.
• To characterize the binding kinetics and potential therapeutic applications of these self-antigen-binding scFv fragments.

Main Methods:

• Phage display library construction using shuffled heavy and light chain V-genes from unimmunized human PBLs.
• Selection of scFv fragments by phage binding to human self-antigens.
• Analysis of scFv fragment composition (monomers/dimers) using FPLC gel filtration.
• Determination of binding kinetics (association, dissociation, affinity) using surface plasmon resonance.

Main Results:

• Isolation of scFv fragments with high specificity for human self-antigens from the phage library.
• Identification of both unmutated and mutated V-genes encoding these self-reactive scFv fragments.
• Characterization of scFv dimer binding kinetics: association (k(on) = 10^5-10^6 M^-1s^-1), dissociation (k(off) = 10^-2 s^-1), and affinity (Ka = 10^7 M^-1).
• Observed relatively fast dissociation kinetics for the scFv dimers.

Conclusions:

• Phage libraries derived from human PBLs can yield scFv fragments with high specificity for self-antigens.
• The binding kinetics, particularly dissociation rates, suggest potential for in vitro affinity maturation for therapeutic purposes.
• Engineered antibody fragments could offer novel therapeutic strategies targeting self-antigens.