Generation of Antibody Libraries for Phage Display: Human Fab Format

Area of Science:

• Molecular Biology
• Immunology
• Biotechnology

Background:

• Phage display is a key technique for generating and refining human monoclonal antibodies.
• The pComb3 phagemid family supports the selection of human antibody libraries in the Fab format.
• Fab format involves fusing human variable domains (V_H, V_L) to constant domains (C_H1, C_L).

Purpose of the Study:

• To describe the utilization of the pC3C phagemid, a pComb3 derivative.
• To generate human Fab libraries with high sequence diversity through random combination of variable domains (V_H, V_κ, V_λ).
• To provide a scalable protocol for library generation from mononuclear cells.

Main Methods:

• Preparation of mononuclear cells from blood and bone marrow.
• Utilizing the pC3C phagemid for creating Fab libraries with randomly combined human variable domains.
• Scaling the protocol to achieve library sizes from 10⁸ to 10¹¹ independent Fab clones.

Main Results:

• Successful generation of human Fab libraries with high sequence diversity.
• The protocol is scalable, accommodating different parental antibody repertoire complexities.
• Demonstrated ability to produce large naive or specialized immune Fab libraries.

Conclusions:

• The pC3C phagemid is effective for generating diverse human Fab libraries.
• This method supports the creation of large naive libraries from healthy donors.
• It also enables the generation of immune libraries from individuals with specific antibody responses.