Contributions of CDR3 to V H H domain stability and the design of monobody scaffolds for naive antibody libraries.
Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.
Bond
Christopher J
CJ
Departments of Medicinal Chemistry, 1 DNA Way, South San Francisco, CA 94080, USA. bond@gene.com
Marsters
James C
JC
Sidhu
Sachdev S
SS
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001703:Biophysics / Q000379:methods; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D016296:Mutagenesis; D019151:Peptide Library; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology
D022801:Complementarity Determining Regions; D015292:Glycoprotein Hormones, alpha Subunit; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D009113:Muramidase; D000409:Alanine