title: Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination.
abstract: Over the past decade, yeast display technology has emerged as a powerful tool for the isolation of high-affinity immunoglobulin fragments with potential utility as clinical diagnostic and therapeutic reagents. Despite significant refinement of the various methodologies underpinning library construction and selections, certain aspects remain challenging and process limiting. We have sought to significantly improve the robustness of the single-chain Fv (scFv) library construction step by overcoming the technical inefficiencies frequently encountered during the PCR-mediated assembly of scFvs from the discrete heavy and light V-domain repertoires. Using a novel primer set designed to provide maximum amplification coverage of the known germ-line V-domain repertoire, we have exploited the potential of the in vivo homologous gap-repair apparatus of Saccharomyces cerevisiae to assemble intact scFvs directly from co-transformed PBMC-derived VH, VL, and linearized vector component fragments. We have successfully applied this three-fragment assembly strategy to construct a large (>10(9)) scFv yeast display library from the ascites immune repertoire of ovarian cancer patients and validated the approach by applying FACS-based sorting to readily isolate scFvs that recognize various tumor marker antigens (TMAs). It is expected that this simplified construction method may find general utility, both for de novo scFv library construction and for subsequent combinatorial affinity maturation manipulations that require more than two fragments.
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.|Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, GuangDong, 510282, China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Yuan
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Mingjuan
authors.initials: M
authors.lastname: Yang
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Hu
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Tingtao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Qirui
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Xuhua
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA.
authors.forename: Ruihe
authors.initials: R
authors.lastname: Lin
authors.affiliation: Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA. zhaoaizhi@gmail.com.
authors.forename: Aizhi
authors.initials: A
authors.lastname: Zhao
mesh_terms: D000951:Antigens, Neoplasm / Q000032:analysis*; D001201:Ascites; D001402:B-Lymphocytes / Q000276:immunology; D061505:Cell Surface Display Techniques / Q000379:methods*; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region; D010051:Ovarian Neoplasms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Scfv; Transformation-associated recombination; Tumor marker antigens; Yeast display library