Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Rabbit monoclonal antibodies offer high affinity and specificity, recognizing conserved epitopes.
• Phage display is a versatile tool for antibody engineering, including de novo generation and humanization.
• Chimeric rabbit/human Fab formats, utilizing rabbit variable domains and human constant domains, enhance expression and purification.

Purpose of the Study:

• To describe a protocol for generating diverse chimeric rabbit/human Fab libraries using a pComb3 derivative (phagemid pC3C).
• To enable the creation of large-scale antibody libraries from rabbit immune or naive repertoires.

Main Methods:

• Utilized phagemid pC3C, a derivative of the pComb3 family, for phage display.
• Generated chimeric rabbit/human Fab libraries by randomly combining rabbit variable domains (V_H, V_κ, V_λ) with human constant domains (C_H1, C_L).
• Initiated library construction from total RNA extracted from rabbit spleen and bone marrow.

Main Results:

• Developed a scalable protocol for generating antibody libraries with high sequence diversity.
• Achieved library sizes ranging from 10⁸ to 10¹¹ independent chimeric rabbit/human Fab clones.
• Demonstrated the utility of the method for creating specialized immune or large naive rabbit antibody libraries.

Conclusions:

• The described phage display method using phagemid pC3C is effective for generating extensive and diverse chimeric rabbit/human Fab libraries.
• This approach facilitates the development of rabbit monoclonal antibodies for research, diagnostics, and therapeutics.
• The protocol's scalability supports the creation of tailored antibody libraries based on specific repertoire needs.