title: Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition.
abstract: Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.
authors.affiliation: Department of Biological Sciences, Florida International University, Miami, FL 33199, USA. jcalmagro@hotmail.com
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Almagro
authors.forename: Veronica
authors.initials: V
authors.lastname: Quintero-Hernández
authors.forename: Mauricio
authors.initials: M
authors.lastname: Ortiz-León
authors.forename: Alvaro
authors.initials: A
authors.lastname: Velandia
authors.forename: Sylvia L
authors.initials: SL
authors.lastname: Smith
authors.forename: Baltazar
authors.initials: B
authors.lastname: Becerril
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000916:Antibody Diversity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D010455:Peptides / Q000276:immunology; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides; C421864:hen egg lysozyme; D009113:Muramidase