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Ciara M Mahon,
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
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ABSTRACT: We have generated large libraries of scFv (>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 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 which 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) and others 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 DLL4. Leads exhibited nM binding affinities, highly specific staining of DLL4+ cells and biochemical neutralisation of DLL4-NOTCH1 interaction.
Journal of Molecular Biology 02/2013; · 4.00 Impact Factor
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Lauren E Smith,
Kathryn Crouch,
Wei Cao,
Mischa R Müller,
Leeying Wu,
John Steven,
Michael Lee,
Musen Liang,
Martin F Flajnik,
Heather H Shih,
Caroline J Barelle, Janet Paulsen,
Davinder S Gill,
Helen Dooley
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ABSTRACT: The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.
Developmental and comparative immunology 04/2012; 36(4):665-79. · 3.29 Impact Factor
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Leeying Wu,
Katarzyna Oficjalska,
Matthew Lambert,
Brian J Fennell,
Alfredo Darmanin-Sheehan,
Deirdre Ní Shúilleabháin,
Bénédicte Autin,
Emma Cummins,
Lioudmila Tchistiakova,
Laird Bloom, Janet Paulsen,
Davinder Gill,
Orla Cunningham,
William J J Finlay
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ABSTRACT: Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.
The Journal of Immunology 11/2011; 188(1):322-33. · 5.79 Impact Factor
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Wei Cao,
Valerie Calabro,
Adam Root,
Grace Yan,
Khetemenee Lam,
Stephane Olland,
Jocelyn Sanford,
Angela Robak,
Richard Zollner,
Zhijian Lu,
Mostafa Ait-Zahra,
Rita Agostinelli,
Lioudmila Tchistiakova,
Davinder Gill,
Douglas Harnish, Janet Paulsen,
Heather H Shih
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ABSTRACT: LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.
FEBS Journal 08/2009; 276(17):4909-20. · 3.79 Impact Factor
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William J Finlay,
Orla Cunningham,
Matthew A Lambert,
Alfredo Darmanin-Sheehan,
Xuemei Liu,
Brian J Fennell,
Ciara M Mahon,
Emma Cummins,
Jason M Wade,
Cliona M O'Sullivan,
Xiang Yang Tan,
Nicole Piche,
Debra D Pittman, Janet Paulsen,
Lioudmila Tchistiakova,
Sreekumar Kodangattil,
Davinder Gill,
Simon E Hufton
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ABSTRACT: Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.
Journal of Molecular Biology 04/2009; 388(3):541-58. · 4.00 Impact Factor
