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Randomized Controlled Trial
. 2013 May 27;425(10):1712-30.
doi: 10.1016/j.jmb.2013.02.015. Epub 2013 Feb 19.

Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential

Ciara M Mahon  1 , Matthew A Lambert, Jacob Glanville, Jason M Wade, Brian J Fennell, Mark R Krebs, Douglas Armellino, Sharon Yang, Xuemei Liu, Cliona M O'Sullivan, Benedicte Autin, Katarzyna Oficjalska, Laird Bloom, Janet Paulsen, Davinder Gill, Marc Damelin, Orla Cunningham, William J J Finlay
Affiliations
Randomized Controlled Trial

Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential

Ciara M Mahon et al. J Mol Biol. .

Abstract

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

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