By-passing immunization. Human antibodies from V-gene libraries displayed on phage

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Traditional antibody production relies on animal immunization and hybridoma technology, which can be time-consuming and inefficient.
• Generating diverse human antibody libraries is crucial for developing targeted therapeutics and research tools.

Purpose of the Study:

• To develop a method for producing human antibodies in bacteria using phage display technology.
• To create and select functional single-chain variable fragment (scFv) antibodies against specific antigens.

Main Methods:

• Constructed diverse libraries of human immunoglobulin variable genes (VH, Vκ, Vλ) from peripheral blood lymphocytes using polymerase chain reaction (PCR).
• Created combinatorial libraries of scFv fragments by random assembly of VH and V-light chain genes.
• Selected antigen-binding phage through iterative panning and growth with specific antigens (turkey egg-white lysozyme, bovine serum albumin) or haptens (2-phenyloxazol-5-one).
• Sequenced selected V-genes and produced soluble antibody fragments for affinity analysis.

Main Results:

• Generated a phage display library with over 10^7 members encoding human antibody fragments.
• Successfully selected and sequenced antibody genes, identifying both germline and mutated variants.
• Demonstrated specific antigen and hapten binding by soluble antibody fragments with high affinities (Ka for TEL = 10^7 M^-1; Ka for phOx = 2 x 10^6 M^-1).

Conclusions:

• Phage display technology enables the isolation of human antibodies against diverse antigens without immunization.
• This method offers a powerful alternative to hybridoma technology for antibody discovery and development.
• Further optimization, such as using larger libraries, may yield even higher-affinity antibodies.