Massively parallel interrogation and mining of natively paired human TCRαβ repertoires

Abstract

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαβ–Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

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Fig. 1: Overview of the massively parallel TCRαβ repertoire mining technology.
Fig. 2: Bioinformatic analysis of six paired TCRαβ repertoires.
Fig. 3: Identification of virus-specific TCRαβ clones by library panning and cellular activation screens.
Fig. 4: Experimental validation of viral-specific TCRαβ clones.
Fig. 5: Identification and experimental validation of antitumor antigen TCRαβ clones from TILs.

Data availability

Presort TCRα–TCRβ repertoire fastq sequence files are deposited at the Sequence Read Archive (http:www.ncbi.nlm.nih.gov/sra/), under BioProject ID PRJNA541985. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was partially funded by National Institute of Allergy and Infectious Diseases grant R43AI120313-01 to D.S.J. and National Cancer Institute grant R43CA232942 to M.J.S. N.O. is supported with funding from the Medical Research Council, Engineering and Physical Sciences Research Council Centre for Doctoral Training (EP/L014904/1). R. Mizrahi, M. Adams, R. Leong and J. Leong (GigaGen) assisted with the development of Gibson assembly protocols. E. Stone and S. Keating (GigaGen) provided useful discussions about general T cell immunology. R. Guest (Immetacyte) provided assistance with isolation and expansion of TILs.

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Contributions

Conceptualization: M.J.S., J.S.B., A.S.A., E.H.M., R.E.H., M.C. and D.S.J.; methodology: M.J.S., E.K.W., J.M.H., N.O., J.S.B., A.S.A., M.A.A., E.H.M. and D.S.J.; software: R.C.E. and Y.W.L.; validation: M.J.S., A.L.N. and E.K.W.; investigation: M.J.S., A.L.N., N.O., J.S.B. and E.K.W.; data curation: M.J.S., E.K.W., J.M.H., Y.W.L., A.S.A. and D.S.J.; writing—original draft preparation: M.J.S. and D.S.J.; writing—review and editing: M.J.S., E.K.W., N.O., J.S.B., J.M.H., A.S.A., Y.W.L., E.H.M., M.C. and D.S.J.; visualization: M.J.S., E.K.W., J.M.H., Y.W.L., A.S.A. and D.S.J.; supervision: M.J.S., J.S.B., A.S.A. and D.S.J.; project administration: M.J.S. and D.S.J.; funding acquisition: M.J.S. and D.S.J.

Corresponding author

Correspondence to David S. Johnson.

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Competing interests

M.J.S., A.L.N., E.K.W., A.S.A., M.A.A., Y.W.L., R.C.E. and D.S.J. are salaried employees of GigaGen, which is an affiliate of GigaMune. GigaMune pays cash to GigaGen for research services. M.J.S., A.L.N., E.K.W., A.S.A., M.A.A., R.C.E., Y.W.L., E.H.M., M.C. and D.S.J. are holders of equity shares in GigaMune. J.M.H. and M.C. hold research positions at the Massachusetts General Hospital. Massachusetts General Hospital has entered into a research collaboration with GigaMune. M.C. is currently an employee of AstraZeneca. M.C. owns equity in Revitope Oncology and Gritstone Oncology. M.C. received consultant fees from Merck Laboratories. J.S.B. and R.E.H. are salaried employees of Immetacyte. R.E.H. is a holder of equity shares in Immetacyte. The viral TCRs and TCR repertoire mining methods are described in US Patent and Trademark Office (USPTO) provisional patent application 62/821808, assigned to GigaMune (M.J.S., A.L.N., E.K.W., Y.W.L., A.S.A., M.A.A. and D.S.J.). The PMEL TCRs are described in USPTO provisional patent application 62/842691, assigned to GigaMune (M.J.S., A.S.A., M.A.A. and D.S.J.). Methods for generating TCR libraries are described in patents WO2012083225A2, US20160362470A1, US20170247684A1 and US20170247683A1, assigned to GigaGen or GigaMune (M.J.S., A.S.A., E.H.M. and D.S.J.).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–41.

Reporting Summary

Supplementary Table 1

Summary of HLA types, CMV and EBV status, number of input cells, number of sequencing reads and number of clonotypes for each of the seven libraries.

Supplementary Table 2

Summary of sequencing data for primary T cells, linked TCR libraries, cloned TCR libraries and Jurkat TCR libraries for each of the seven libraries generated in the study.

Supplementary Table 3

Summary of the MHC dextramers used in the Jurkat panning experiments.

Supplementary Table 4

CDR3, V-gene and J-gene sequences for each TCR identified in the Jurkat panning experiments, plus their frequencies in the initial primary T cell sequencing data.

Supplementary Table 5

CDR3, V-gene and J-gene sequences for each TCR identified in the Jurkat panning experiments, plus their frequencies in the initial primary T cell sequencing data and at each step of the Jurkat panning experiments. Also included are MHC dextramer-binding and Jurkat activation validation data for monoclonal TCR constructs.

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Spindler, M.J., Nelson, A.L., Wagner, E.K. et al. Massively parallel interrogation and mining of natively paired human TCRαβ repertoires. Nat Biotechnol 38, 609–619 (2020). https://doi.org/10.1038/s41587-020-0438-y

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