The development of a canine single-chain phage antibody library to isolate recombinant antibodies for use in translational cancer research

Area of Science:

• Immunology and Biotechnology
• Canine and Comparative Medicine

Background:

• Developing immunotolerant monoclonal antibodies is crucial for advancing veterinary and human medicine.
• Canine models offer unique insights into human disease pathogenesis and treatment.

Purpose of the Study:

• To establish a robust methodology for cloning and archiving the canine variable domain antibody repertoire.
• To create a synthetic antibody platform for developing novel therapeutics and preclinical models.

Main Methods:

• Utilized 5'RACE PCR to clone naive, somatically mutated variable domain repertoire from canine B cell mRNA.
• Developed degenerate primers for cloning variable domain genes into archival plasmid libraries.
• Constructed a single-chain variable fragment (scFv) M13 phage library through combinatorial ligation.
• Employed next-generation sequencing (long-read and short-read) for library annotation, including CDR diversity and immunoglobulin gene subfamily distribution.

Main Results:

• Successfully cloned and archived a diverse canine antibody variable domain repertoire.
• Annotated library features, revealing CDR diversity and IGHV/IGKV/IGLV subfamily distribution.
• Generated a synthetic immunoglobulin G targeting the canine immune checkpoint receptor PD-1.

Conclusions:

• The developed synthetic platform enables perpetual cloning and annotation of antibody variable domain genes.
• This platform facilitates the development of improved preclinical models for complex human diseases.
• The methodology accelerates the discovery of novel canine and human therapeutics.