Abstract

CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. The raw data for Figs. 15 and Supplementary Figs. 1, 3, 4, 7, and 8 can be found in the Source Data files. The list of epitopes in the SABR libraries can be found in Supplementary Tables 3 and 4. The plasmids for HLA-A*0201-SABR backbone (pCCLc-MND-A0201-SABR-Backbone; ID 119050), HLA-B*2705-SABR backbone (pCCLc-MND-B2705-SABR-Backbone; ID 119051), A2-Mart1-SABR (pCCLc-MND-A0201-Mart1-SABR; ID 119052), and B27-KK10-SABR (pCCLc-MND-B2705-KK10-SABR; ID 119053) are available through Addgene. The sequencing data have been deposited in the Sequence Read Archive (SRR8207921, amplicon sequencing of A2-SABR-library co-incubated with F5 TCR; SRR8207922, amplicon sequencing of A2-SABR-library co-incubated with SL9 TCR; SRR8207923, amplicon sequencing of A2-SABR-library co-incubated with no TCR; SRR8207924, amplicon sequencing of A2-NeoAg-library co-incubated with neoTCR; SRR8207925, amplicon sequencing of A2-NeoAg-library co-incubated with no TCR). The code used to analyze sequences has been deposited in GitHub (https://github.com/Baltimore-Lab/nat-methods-SABR-trogo).

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Acknowledgements

We thank I. Antoshechkin at the Millard and Muriel Jacobs Genetics and Genomics Laboratory for Illumina sequencing, and A. Spalla at the Analytical Cytometry Core at the City of Hope for help with FACS. NFAT-GFP-Jurkat cells were a gift from A. Weiss (University of California, San Francisco, San Francisco, CA, USA) and Y. Chen (University of California, Los Angeles, Los Angeles, CA, USA). GXR-B27+ cells were a gift from B.D. Walker (Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Cambridge, MA, USA). J3 chimeric antigen receptor was a gift from P. Wang (University of Southern California, Los Angeles, CA, USA). The pCCLc-MND-X backbone and pCMV-RD8.9 were gifts from D.B. Kohn (University of California, Los Angeles, Los Angeles, CA, USA). MSCV-based shuttle plasmid was a gift from R.A. Morgan (National Institutes of Health, Bethesda, MD, USA). This work was funded by the California Institute for Regenerative Medicine (award DISC2-09123 to D.B.), the Caltech Rothenberg Innovation Initiative (to D.B.), and the US National Cancer Institute (grant 1U54 CA199090-01 to J.R.H.).

Author information

Affiliations

  1. Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    • Alok V. Joglekar
    • , Michael T. Leonard
    • , John D. Jeppson
    • , Margaret Swift
    • , Guideng Li
    • , Stephanie Wong
    • , Michael T. Bethune
    •  & David Baltimore
  2. Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    • Guideng Li
  3. Suzhou Institute of Systems Medicine, Suzhou, China

    • Guideng Li
  4. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    • Songming Peng
    •  & James R. Heath
  5. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    • Jesse M. Zaretsky
    •  & Antoni Ribas
  6. Parker Institute for Cancer Immunotherapy (PICI), University of California, Los Angeles and California Institute of Technology, Pasadena, CA, USA

    • James R. Heath
    • , Antoni Ribas
    •  & David Baltimore
  7. Division of Hematology & Oncology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    • Antoni Ribas
  8. Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    • Antoni Ribas

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Contributions

A.V.J. designed and performed experiments, analyzed and interpreted the data, and wrote the manuscript. M.T.L. designed and performed experiments, performed computational analyses, and interpreted the data. M.S. and J.D.J. designed and performed experiments, and analyzed the data. G.L., S.W., S.P., J.M.Z., and M.T.B. designed and performed experiments, and contributed reagents. J.R.H. and A.R. contributed reagents and supervised experiments. D.B. supervised the experiments, analyzed and interpreted the data, and wrote the manuscript.

Competing interests

A.V.J., M.T.L., M.T.B., and D.B. are named as co-inventors on a patent application concerning the described technology. D.B. is a consultant of PACT and head of their scientific advising board. J.R.H. and A.R. are directors and consultants of PACT; M.T.B. and S.P. are employees of PACT; J.M.Z. is a consultant of PACT; and each of the foregoing individuals has equity interests in PACT.

Corresponding authors

Correspondence to Alok V. Joglekar or David Baltimore.

Integrated supplementary information

  1. Supplementary Figure 1 Variants of SABR constructs.

    a. Schematics showing the SABR-F and SABR-E constructs. SABR-F contains the transmembrane domain from HLA (black rectangle), whereas SABR-E contains the transmembrane domain from CD3ζ (teal rectangle). The two horizontal lines indicate two leaflets of the plasma membrane. b. Representative flow cytometry plots showing GFP expression in coculture assays from Fig 1b. The experiments were performed at n = 3 biologically independent cell culture replicates. c. GFP expression in coculture assays comparing SABR-F and SABR-E. The lines and error bars indicate mean ± s.d. from n = 12 biologically independent cell culture replicates. Source Data

  2. Supplementary Figure 2 Time course of GFP expression induced by SABRs.

    Representative flow cytometry plots from the F5+A2-MART1 experiment enumerated in Fig 1e are shown. The rectangle in the right bottom corners shows the gate for counting GFP+ cells. The time at which each sample was collected is shown as hours. The frequency of cells in the GFP+ gate is indicated as a percentage.

  3. Supplementary Figure 3 Recognition of low-affinity TCRs by SABRs.

    GFP expression in coculture assays using F5 and M1 TCRs with A2-MART1-SABR. The dots indicate individual values from n = 2 biologically independent cell culture replicates. Source Data

  4. Supplementary Figure 4 Empty SABR vector constructs and recognition of low-affinity pMHC–TCR interactions.

    a. Schematic showing SCT, SABRs, empty SABRs, and TMGs. EP, epitope; S, signal sequence; MITD, MHC class I trafficking signal; numbers 1–6 indicate Gly–Ser linkers. b. Correlation of functional avidity of interaction of EC27 TCR with variants of KK10 peptides with their ability to initiate signal through SABRs. The indicated peptides are variants of the KK10 epitope. R2T, KTWIILGLNK; R2I, KIWIILGLNK; R2G, KGWIILGLNK; R2K, KKWIILGLNK; WT, KRWIILGLNK; R2Q, KQWIILGLNK. Source Data

  5. Supplementary Figure 5 Strategy to clone custom oligonucleotides into SABR vectors.

    SABR vector constructs with a stuffer fragment showing BsmBI sites (top), and cloning strategy using double-stranded oligonucleotides with encoding the epitope flanked by overlaps.

  6. Supplementary Figure 6 SABR library screen for antigen discovery.

    a. Schematic showing the pipeline to construct custom SABR libraries. EP, epitope. The left panel shows the procedure to obtain and synthesize a list of epitopes. The right panel shows the schematic of the SABR library. b. Schematic showing coculture experiment to select cells from SABR library that are recognized by an orphan TCR. Left panel shows a SABR library presenting numerous unique epitopes. The middle panel shows antigen-presenting cells (APCs) showing reporter expression induced by SABRs presenting the cognate epitope for the orphan TCR. Right panel shows processing of the selected cells. c. Flowchart showing the computational analysis pipeline.

  7. Supplementary Figure 7 Enrichment of EAAGIGILTV and SLYNTVATL analogs in SABR library screen.

    a. Average ranks for all the EAAGIGILTV analogs in the A2-SABR library. b. Average ranks for all the SLYNTVATL analogs in the A2-SABR library. The ranks were calculated as described in the manuscript. The data are averaged from three biologically independent cell culture replicates. Source Data

  8. Supplementary Figure 8 Enrichment of USP7 neoepitopes in SABR library screen.

    Average ranks for all the USP7-derived epitopes in the NeoAg-SABR library. The data are averaged from three biologically independent cell culture replicates. Source Data

  9. Supplementary Figure 9 Gating strategy used in flow cytometry.

    a. Gating strategy used in coculture assays to measure GFP expression. b. Gating strategy used in coculture assays to measure GFP and CD69 expression. c. Gating strategy used in cytotoxicity assays.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figs. 1–9 and Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Protocol

    A cell-based platform for T cell antigen discovery: engineered antigen-presenting cells expressing signaling and antigen-presenting bifunctional receptors (SABRs).

  4. Supplementary Table 3

    The list of epitopes in the A2-SABR library.

  5. Supplementary Table 4

    The list of epitopes in the A2-NeoAg library.

Source Data

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https://doi.org/10.1038/s41592-018-0304-8