title: Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling.
abstract: Light (L) chain shuffling is routinely used to analyze optimal L chains that pair with a specific heavy (Fd or H) chain, which ultimately leads to in vitro affinity maturation of a particular antibody. One of the major drawbacks to this procedure is that L chain libraries have to be created for each distinct H chain, which involves complicated cloning procedures. Herein, we designed of the dual-vector system-III (DVS-III), which is composed of a set of pLf1T-3 phagemid and pHg3A-3 plasmid, for L chain shuffling of any given human Fab antibody via phage display technology. To demonstrate the feasibility of our system, a human naïve L chain sublibrary, HuNL-D3, constructed in pLf1T-3 phagemid, was combined with the Fd of a human anti-IL-15 Fab, 4H10, subcloned in pHg3A-3 plasmid as a model system. After solution-phase sorting and biopanning the library we obtained eight Fab variants (4H10-LP1-7 and 4H10-LS). Among them, 4H10-LP4 exhibited the highest affinity which is about 36-fold higher than that of the parent molecule 4H10 (K(D)=6 nM versus 200 nM). Our results demonstrate that the DVS-III, along with the HuNL-D3 L chain sublibrary, can be served as a convenient approach for affinity maturation of any given human Fab antibody through L chain optimization.
authors.affiliation: Division of Molecular and Medical Biotechnology, College of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea.
authors.forename: Byung-ung
authors.initials: BU
authors.lastname: Hur
authors.forename: Hyo-jung
authors.initials: HJ
authors.lastname: Choi
authors.forename: Suk-Yoon
authors.initials: SY
authors.lastname: Song
authors.forename: Jae-bong
authors.initials: JB
authors.lastname: Yoon
authors.forename: Li-kun
authors.initials: LK
authors.lastname: Liu
authors.forename: Sang-hoon
authors.initials: SH
authors.lastname: Cha
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library