(The) versatility of framework regions of chicken VH and VL to mutations

Abstract

Introduction: Diversification of chicken immunoglobulin occur by somatic recombination and hypermutation during immune responses. Different to mutations in framework regions, mutations in Complemenatarity determining regions may be positively selected by antigen, leading to enhanced binding but not in framework regions.

Aim: To select replaceable amino acids from the randomized chicken antibody framework residues and replacing part of them with cysteine for PEGlytion.

Methods: Reactivity of anti-PSA monoclonal antibody selected from chicken antibody library and each of its framework regions randomized to antigen was measured by enzyme-linked immunosorbent assay. In addition, we conjugated Polyethylene glycol to cysteine replaced scFvs. Lastly, we applied next-generation sequencing analysis to select rare and diverse scFvs by using phagemid DNA which were prepared after bio-panning.

Results: Following the results, most residues could be replaced to other residues while retaining high affinity to antigen. Although framework region could be replaced with various amino acids, the somatic mutation in framework may be lost during clonal expansion because of negative mutations. In addition, three VL (L5, L6 and L7) and two VH (H13 and H16) cysteine mutants as scFv-Ckappa fusion proteins showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all the five mutants. Furthermore, we selected various scFvs by NGS analysis.

Conclusions: Therefore, chicken antibody could be useful to select diverse antibodies with antigen binding activity and the replacement of framework of chicken antibody may be useful for an approach to chicken antibody humanization, affinity maturation and drug conjugation.