title: Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential.
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.
authors: Pfizer, Global Biotherapeutics Technologies, Grange Castle Business Park, Clondalkin, Dublin 22, Ireland.
authors: Ciara M
authors: CM
authors: Mahon
authors: Matthew A
authors: MA
authors: Lambert
authors: Jacob
authors: J
authors: Glanville
authors: Jason M
authors: JM
authors: Wade
authors: Brian J
authors: BJ
authors: Fennell
authors: Mark R
authors: MR
authors: Krebs
authors: Douglas
authors: D
authors: Armellino
authors: Sharon
authors: S
authors: Yang
authors: Xuemei
authors: X
authors: Liu
authors: Cliona M
authors: CM
authors: O'Sullivan
authors: Benedicte
authors: B
authors: Autin
authors: Katarzyna
authors: K
authors: Oficjalska
authors: Laird
authors: L
authors: Bloom
authors: Janet
authors: J
authors: Paulsen
authors: Davinder
authors: D
authors: Gill
authors: Marc
authors: M
authors: Damelin
authors: Orla
authors: O
authors: Cunningham
authors: William J J
authors: WJ
authors: Finlay
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use*; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D051881:Receptor, Notch1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D022801:Complementarity Determining Regions; C424126:DLL4 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C497761:NOTCH1 protein, human; D019151:Peptide Library; D051881:Receptor, Notch1; D057127:Single-Chain Antibodies