Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Abstract

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human D J regions was used to isolate low-affinity antibodies to human β nerve growth factor (hβNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K D . This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs. affinity maturation | mammalian display

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

“…The library was assembled in episomes in a full-length IgG1 format that supports simultaneous secretion and cell surface display in mammalian cells (14).…”

“…Transformation, Stable Expression, and Selection of HEK293 c18 Cells-HEK293 c18 cell lines stably expressing the CDR3-grafted, IgG heavy chains modified with a C-terminal transmembrane domain (22) and CDR1,2,3-grafted light chain, together with AID were generated using individual episomal vectors under independent selection for expression of HC, LC, and AID as described (14). T75 culture flasks were seeded with 3 ϫ 10 6 HEK293 c18 cells in DMEM containing 10% FBS (Invitrogen).…”

“…Affinity Maturation-APE424 was affinity-matured using two parallel strategies: in vitro SHM initiated by transfection of AID and small combinatorial libraries incorporating the most frequent in vivo SHM events in the LC CDR1 and 2. In the first approach, the HC and LC were stably transfected with AID into HEK293 cells as described (14). In the second approach, positions within the LC CDR1 and CDR2 were varied in combinatorial libraries to identify beneficial mutations.…”

“…Structure-based (5-7) and library-based methods (8) have been demonstrated for antibody humanization that regain or may improve upon the binding affinity of the originating molecule. Affinity maturation of antibodies can be accomplished by a number of methods including random mutagenesis (9,10), random mutagenesis of CDR sequences (11), directed mutagenesis of residues (12,13), and approaches that reproduce SHM in vitro (14). Random methods for humanization and affinity maturation rely on multiple rounds of labor-intensive trial and error mutagenesis, and may result in an additional non-germ line sequence being incorporated into the final antibody.…”

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

“…Recently, a mammalian cell surface antibody display system has offered one new approach, but this technology depends on the transient transfection of expression plasmids (9,10). A eukaryotic display technology that also has a virus display platform as well as cell surface display capabilities would be ideal.…”