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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

  • Applied genetics and molecular biotechnology
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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 (>109) 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.

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Acknowledgments

This research was supported by the Honorable Tina Brozman Foundation (AZ), the Claneil Foundation (AZ), and the NCI FCCC-UPenn Cancer SPORE pilot grant (AZ).

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Authors and Affiliations

  1. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19014, USA

    Xiaopeng Yuan, Xiang Chen, Mingjuan Yang, Jia Hu, Wei Yang, Tingtao Chen, Qirui Wang, Xuhua Zhang, Ruihe Lin & Aizhi Zhao

  2. Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, GuangDong, 510282, China

    Xiaopeng Yuan

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  1. Xiaopeng Yuan
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  2. Xiang Chen
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Correspondence to Aizhi Zhao.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Xiaopeng Yuan, Xiang Chen, and Mingjuan Yang contribute equally.

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Yuan, X., Chen, X., Yang, M. et al. 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. Appl Microbiol Biotechnol 100, 4051–4061 (2016). https://doi.org/10.1007/s00253-016-7303-4

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