Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing.
The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-β3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-β3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-β3 binding and neutralized TGF-β3 in in vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.
Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, CanadaK1A 0R6.
Kevin A
KA
Henry
Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, CanadaK1A 0R6.
Greg
G
Hussack
Human Health Therapeutics Portfolio, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, CanadaH4P 2R2.
Cathy
C
Collins
Human Health Therapeutics Portfolio, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, CanadaH4P 2R2.
John C
JC
Zwaagstra
Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6.|School of Environmental Sciences, Ontario Agricultural College, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 75 Laurier Avenue East, Ottawa, Ontario, Canada K1H 8M5.
Jamshid
J
Tanha
Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6.|School of Environmental Sciences, Ontario Agricultural College, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.
C Roger
CR
MacKenzie
D000818:Animals; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D002161:Camelids, New World / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D055550:Protein Stability; D017422:Sequence Analysis, DNA; D061905:Single-Domain Antibodies / Q000235:genetics* / Q000276:immunology*; D016212:Transforming Growth Factor beta / Q000276:immunology*
D057134:Antibodies, Neutralizing; D000939:Epitopes; D061905:Single-Domain Antibodies; D016212:Transforming Growth Factor beta
TGF-β; VHH; antibody; next-generation DNA sequencing; phage display; single-domain antibody