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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The data that support the findings of this study are available from the corresponding author upon request. The raw data for Figs. 1–5 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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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.).
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.
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Integrated supplementary information
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.
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.
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.
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.
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.
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.
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.
Average ranks for all the USP7-derived epitopes in the NeoAg-SABR library. The data are averaged from three biologically independent cell culture replicates.
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 Figs. 1–9 and Supplementary Tables 1 and 2
A cell-based platform for T cell antigen discovery: engineered antigen-presenting cells expressing signaling and antigen-presenting bifunctional receptors (SABRs).
The list of epitopes in the A2-SABR library.
The list of epitopes in the A2-NeoAg library.
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Joglekar, A.V., Leonard, M.T., Jeppson, J.D. et al. T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nat Methods 16, 191–198 (2019). https://doi.org/10.1038/s41592-018-0304-8
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