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Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening

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Abstract

Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

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Acknowledgements

The authors thank the Portuguese Funding Agency, Fundacão para a Ciência e a Tecnologia, FCT IP, for financial support: PTDC/QEQ-MED/4412/2014 to J.G, IF/01010/2013 to FAS.

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Authors and Affiliations

  1. iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Av. Professor Gama Pinto, 1649-019, Lisbon, Portugal

    Joana H. Ministro, Soraia S. Oliveira, Joana G. Oliveira, Miguel Cardoso, Sofia Corte-Real & Joao Goncalves

  2. Technophage SA, Av. Prof. Egas Moniz, Edifício Egas Moniz, 1649-028, Lisbon, Portugal

    Joana H. Ministro, Soraia S. Oliveira, Joana G. Oliveira & Sofia Corte-Real

  3. CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477, Lisbon, Portugal

    Frederico Aires-da-Silva

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  1. Joana H. Ministro
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  2. Soraia S. Oliveira
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  3. Joana G. Oliveira
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Correspondence to Joao Goncalves.

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Conflict of interest

This study was supported by TechnoPhage SA. The authors would like to disclose that TechnoPhage SA have a pending patent application related to the development of CellectAb (patent number 9487773). JM, SO, JO, and SCR at the time of the work were employees of TechnoPhage SA and therefore have a theoretical conflict of interest through being employed by the organization that both funded the work and has potential commercial interest in the findings. JG, FAS, and MC have no financial interests in TechnoPhage.

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Ministro, J.H., Oliveira, S.S., Oliveira, J.G. et al. Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening. Appl Microbiol Biotechnol 104, 2501–2512 (2020). https://doi.org/10.1007/s00253-020-10423-3

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