Combinatorial mutagenesis with alternative CDR-L1 and-H2 loop lengths contributes to affinity maturation of antibodies

Loop length variation in the complementary determining regions (CDRs) 1 and 2 encoded in germline variable antibody genes provides structural diversity in naive antibody libraries. In synthetic single framework libraries the parental CDR-1 and CDR-2 length is typically unchanged and alternative lengths are provided only at CDR-3 sites. Based on an analysis of the germline repertoire and structure-solved anti-hapten and anti-peptide antibodies, we introduced combinatorial diversity with alternative loop lengths into the CDR-L1, CDR-L3 and CDR-H2 loops of anti-digoxigenin and anti-microcystin-LR single chain Fv fragments (scFvs) sharing human IGKV3-20/IGHV3-23 frameworks. The libraries were phage display selected for folding and affinity, and analysed by single clone screening and deep sequencing. Among microcystin-LR binders the most frequently encountered alternative loop lengths were one amino acid shorter (6 aa) and four amino acids longer (11 aa) CDR-L1 loops leading up to 17- and 28-fold improved affinity, respectively. Among digoxigenin binders, 2 amino acids longer (10 aa) CDR-H2 loops were strongly enriched, but affinity improved anti-digoxigenin scFvs were also encountered with 7 aa CDR-H2 and 11 aa CDR-L1 loops. Despite the fact that CDR-L3 loop length variants were not specifically enriched in selections, one clone with 22-fold improved digoxigenin binding affinity was identified containing a 2 residues longer (10 aa) CDR-L3 loop. Based on our results the IGKV3-20/IGHV3-23 scaffold tolerates loop length variation, particularly in CDR-L1 and CDR-H2 loops, without compromising antibody stability, laying the foundation for developing novel synthetic antibody libraries with loop length combinations not existing in the natural human Ig gene repertoire.

Automated summary

This study discusses the impact of loop length variation on the structural diversity of antibody libraries. Introducing loop length variation in specific antibody fragments can enhance affinity, especially in CDR-L1 and CDR-H2 loops. The results lay the foundation for developing novel synthetic antibody libraries with unique loop length combinations.