Fig. 1.
Schematic of ABS library design, selection and screening principles. (A) Amino acid sequences are shown for the v-domains of Par-RAGE and destination germ-line (DPK9-DP54) scFvs in VL-VH format. Rat parental antibody-specific residues are highlighted in red, and human germ-line-matching residues are highlighted in green. At each position where the rat and human residues differed, both residues were encoded for in the ABS-RAGE library. This principle was applied to all CDRs other than the CDR-H3, in a single combinatorial library. In the CDR-H3, point mutations were permitted at a frequency of 1 ± 1 per clone. (B and C) Phage libraries were generated (B) and used in selections on cognate antigen (C). (D) Selection output clones were subsequently screened by ELISA, HTRF, and DNA sequencing to identify hits with maintained target binding and epitope specificity. (E and F) Top clones were expressed and purified as IgGs (E), before characterization of affinity by Biacore, solubility and aggregation analyses by SEC, in vitro specificity by ELISA and Biacore, and thermal stability analysis by DSC (F).