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 naïve 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.

Next generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections.

Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2×300bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening.