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Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting

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Abstract

Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:108 by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 106 negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

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Acknowledgments

We thank Chunchun Liu and Junying Jia for their technical assistance in flow cytometry analysis and sorting. This work was supported by National Science and Technology Major Project (2013ZX10004611-002 and 2013ZX10003006-001-001), National Special Projects for Key Scientific Instruments and Equipment Development of Ministry of Science and Technology (2011YQ03013404), and State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences (SKLMR-20130605). It was also funded by National Natural Science Funds of China (31500753 and 31400041).

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Authors and Affiliations

  1. Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Shuang Sun, Yun Zhao, Yan Lin, Chen Ye & Haiying Hang

  2. University of Chinese Academy of Sciences, Beijing, 100039, China

    Shuang Sun

  3. CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China

    Xiao Yang & Xiangdong Fang

  4. College of Life Sciences, North China University of Science and Technology, Tangshan, Hebei Province, 063000, China

    Haifeng Wang

  5. Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Haiying Hang

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  1. Shuang Sun
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Correspondence to Haiying Hang.

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Shuang Sun and Xiao Yang contributed equally to the work.

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Sun, S., Yang, X., Wang, H. et al. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting. Appl Microbiol Biotechnol 100, 5977–5988 (2016). https://doi.org/10.1007/s00253-016-7472-1

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