Platform for high-throughput antibody selection using synthetically-designed antibody libraries

Abstract

Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>1020), the practical diversity that can be achieved by transformation of E. coli is limited to about 1010. To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

Rational library design by functional CDR resampling

“…Based on this hypothesis, such a library was designed, constructed and validated. To achieve maximum compatibility, we utilized a legacy database consisting of ~2000 sequences of scFv domains, discovered in a single-framework library generated via a traditional degenerate method, where “NNK” codons were substituted at ~15 different positions on a single scFv template; the parental scFv was developed by linking the VL and VH domains of a human anti-IFNα antibody with a 12 amino acid glycine linker [16].…”

“…Therefore, we picked a set of 12 folded proteins and 8 synthetic peptides (proteins: hIL-12p70, FGF basic protein, Dtk-Fc, BCMA-Fc, Nogo Fc, IL-13, myelin basic protein, hLeptin, IFN-α, BDNF, IL-1 beta, IL3-β;,peptides: XIAP1B, XIAP2B, GRAP2B, GRAP3B, LMNA3B, TDP_42_NLS, XIAP3B, LMNA2B) as the initial set to screen with the two PDC generated libraries. The phage display discovery screening was carried out following previously published protocols [16]. Hit sequences were analyzed by a bioinformatics pipeline written in Python/R, where each hit sequence can be uniquely identified by four integers as the entry ID stored in our CDR sequence database (Figure 5).…”

“…The phage display discovery screening was carried out following previously published protocols [16]. Three rounds of enrichment were performed.…”

“…“NMY” or “NNK”), excluding stop codons, at naturally diversified amino acid sites within selected complementarity determining regions (CDRs) [14]. More advanced methods using defined CDR sequences [15] or triplet codon synthesis to bias towards non-rare codons, as well as to imitate the natural distribution of amino acids at multiple sites of the CDR, have also been used [16,17]. Moreover, randomizing the combination of naïve light chain and heavy chain variable domains has been tested ti diversify a library at a level higher than primary sequences [18].…”

pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

“…To capitalize on the investment and availability of sequenced genomes, it will be essential to devise methods for rapidly and inexpensively generating affinity reagents at a proteomescale. Combining the pMINERVA system with our previous improvements, AXM mutagenesis (55), Phage ESCape (56), and phage libraries using entirely pre-defined complementarity determining regions (CDR) (57) we present a platform for the rapid generation of recombinant monoclonal Abs in as few as three weeks and at a cost comparable to that of polyclonal antibodies. The scFv Abs encoded on the pDonor vector as M13gpIII-fusions can be screened in a phage display biopanning procedure to identify a phagemid encoding a scFv with the desired biophysical properties.…”