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High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

An Author Correction to this article was published on 22 May 2020

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Abstract

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

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Fig. 1: The CelliGO process.
Fig. 2: Phenotypic screening and sorting of IgG-secreting cells.
Fig. 3: Sequencing, expression and characterization of IgGs expressed by sorted cells.

Data availability

Processed sequencing files (quality trimmed, merged and barcode assigned) for all data generated in this study were deposited at Sequence Read Archive (SRA) under accession PRJNA529803.

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Acknowledgements

We would like to thank M. Holsti and W. Somers (Pfizer, BioMedicine Design) for advice on the technology development and critical reading of the manuscript; R. Nicol (Whitehead Institute, MIT Center for Genome Research) for advice on barcoded sequencing; at the Institut Pasteur, H. Mouquet for advice on antibody sequence selection, and F. Jönsson for advice on flow cytometry analysis; T. Kirk and S. Foulon (ESPCI Paris) for their help developing the barcoded hydrogel beads; S. N. Stewart (HiFiBio Therapeutics Inc.) for her help producing and analysing recombinant antibodies; the Institut Pierre-Gilles de Gennes (IPGG) for use of clean room facilities and the laser engraver (CII08, Axyslaser). This work was supported by the French Agence Nationale de la Recherche (ANR-14-CE16-0011 project DROPmAbs), by the Centre d’Innovation et Recherche Technologique (Citech) through the Institut Carnot Pasteur Microbes & Santé (ANR 16 CARN 0023-01), by BPI France (OSIRIS and CELLIGO projects) and by the French ‘Investissements d’Avenir’ program via the CELLIGO project and grant agreements ANR-10-NANO-02, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31 and ANR-10-EQPX-34. NGS was performed by the ICGex NGS platform of the Institut Curie and the Institut de Biologie Intégrative de la Cellule (I2BC) platform (Gif-sur-Yvette) supported by the grants ANR-10-EQPX-03 and ANR-10-INBS-09-08 (France Génomique Consortium), by the Canceropole Ile-de-France and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS- 4654). C.C. acknowledges financial support from CONCYTEC, Peru. P.C.H. is a scholar in the Pasteur - Paris University (PPU) International PhD program and was also supported by the Fondation pour la Recherche Médicale (FRM; FDT201904008240). K.E. acknowledges financial support from the Swiss National Science Foundation and The Branco Weiss Fellowship - Society in Science.

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Authors

Contributions

C.B., P.B., A. Gérard, A.J. and A.D.G. designed and supervised the study; C.B., P.B. and A.D.G. secured funding; L.B.-R., P.C.-H., C.C., M.D., R.D., K.E., S. Elllouze., S. Essono., A. Godina, K.G., B.I., B.J., R.K., V.M., P.M., S.M., G.M., C.O., Y.P., A.P., M.R., O.R.-L., G.R., A.S.-C., B. Saudemont., B. Shen., S.N.S. and A.W. performed the experiments; J.B., C.B., P.B., P.E., A. Gérard, A.D.G., A.J., G.A.M., G.M., C.N. and A.W. analyzed the data, and P.B., A. Gérard, A.D.G. and A.W. wrote the paper.

Corresponding authors

Correspondence to Andrew D. Griffiths, Pierre Bruhns or Colin Brenan.

Ethics declarations

Competing interests

A.D.G. and C.B. are co-founders of Seven Pines Holding BV, and HiFiBiO Therapeutics SAS is a subsidiary of Seven Pines Holding BV. Patents have been filed on some aspects of this work and future development of the platform, and the inventors may receive payments related to exploitation of these under their employer’s rewards to inventors scheme.

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Supplementary information

Supplementary information

Supplementary Figures 1–26, Supplementary Tables 1 and 2, and Supplementary Note 1.

Reporting Summary

Supplementary Video 1

Cell and hydrogel bead co-compartmentalization into ~1 nl droplets. The aqueous phase containing cells and hydrogel beads, as well as lysis and reverse transcription reagents (right) were co-compartmentalized at the T-junction (middle) to form ~1 nl droplets (left) containing reagents necessary to lyse cells and initiate reverse transcription of VH–VL mRNA to form barcoded cDNA.

Supplementary Table 3

Expressed recombinant IgGs tested for binding to TT, GPI or TSPAN8-overexpressing cells

Supplementary Table 4

List of unique VH–VL pairs identified across sorts of splenocytes from TT, GPI and TSPAN8-overexpressing cell immunized mice.

Supplementary Table 5

Sequences of single-cell barcode indexes.

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Gérard, A., Woolfe, A., Mottet, G. et al. High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics. Nat Biotechnol 38, 715–721 (2020). https://doi.org/10.1038/s41587-020-0466-7

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