title: Synthetic antibodies designed on natural sequence landscapes.
abstract: We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(κ)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(κ) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.
authors.affiliation: Rinat, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080, USA.
authors.forename: Wenwu
authors.initials: W
authors.lastname: Zhai
authors.forename: Jacob
authors.initials: J
authors.lastname: Glanville
authors.forename: Markus
authors.initials: M
authors.lastname: Fuhrmann
authors.forename: Li
authors.initials: L
authors.lastname: Mei
authors.forename: Irene
authors.initials: I
authors.lastname: Ni
authors.forename: Purnima D
authors.initials: PD
authors.lastname: Sundar
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Blarcom
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Abdiche
authors.forename: Kevin
authors.initials: K
authors.lastname: Lindquist
authors.forename: Ralf
authors.initials: R
authors.lastname: Strohner
authors.forename: Dilduz
authors.initials: D
authors.lastname: Telman
authors.forename: Guido
authors.initials: G
authors.lastname: Cappuccilli
authors.forename: William J J
authors.initials: WJ
authors.lastname: Finlay
authors.forename: Jan
authors.initials: J
authors.lastname: Van den Brulle
authors.forename: David R
authors.initials: DR
authors.lastname: Cox
authors.forename: Jaume
authors.initials: J
authors.lastname: Pons
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D006801:Humans; D008962:Models, Theoretical; D019151:Peptide Library
chemical_list: D000906:Antibodies; D019151:Peptide Library