Abstract
T cell receptor (TCR) gene therapy is a potent form of cellular immunotherapy in which patient T cells are genetically engineered to express TCRs with defined tumor reactivity. However, the isolation of therapeutic TCRs is complicated by both the general scarcity of tumor-specific T cells among patient T cell repertoires and the patient-specific nature of T cell epitopes expressed on tumors. Here we describe a high-throughput, personalized TCR discovery pipeline that enables the assembly of complex synthetic TCR libraries in a one-pot reaction, followed by pooled expression in reporter T cells and functional genetic screening against patient-derived tumor or antigen-presenting cells. We applied the method to screen thousands of tumor-infiltrating lymphocyte (TIL)-derived TCRs from multiple patients and identified dozens of CD4+ and CD8+ T-cell-derived TCRs with potent tumor reactivity, including TCRs that recognized patient-specific neoantigens.
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Data availability
DNA sequencing data of TCR discovery screens have been deposited in the National Center for Biotechnology Informationʼs Sequence Read Archive under accession codes PRJNA1068078 (ref. 41), PRJNA1068299 (ref. 42), PRJNA1068301 (ref. 43) and PRJNA1068303 (ref. 44).
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Acknowledgements
We would like to thank P. Kaptein and J. Urbanus for valuable help with developing the screening technology; S. Ketelaars for bioinformatic support; R. Tissier for support with statistical analyses; the Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital (NKI-AVL) Flow Cytometry Facility for flow cytometric support; the NKI-AVL Core Facility Molecular Pathology & Biobanking for supplying NKI-AVL Biobank material and laboratory support; and the NKI-AVL Genomics Core Facility for support with next-generation sequencing. This work was supported by the Dutch Cancer Society Young Investigator Grant (no. 2020-1/12977) (to W.S.); ZonMw Translational Research Program 2 (no. 446002001) (to W.S. and J.B.A.G.H.); the Stevin Prize and the Louis-Jeantet Prize (to T.N.S.); National Institutes of Health grants (no. R01CA269898 and no. R37CA273333-01) (to J.W.); the V Foundation (to J.W.); the Mark Foundation ASPIRE award (to J.W.); the Melanoma Research Alliance Young Investigator award (to J.W.); and a generous donation from Florry Vyth (to J.B.A.G.H.). Research at the Netherlands Cancer Institute is supported by institutional grants from the Dutch Cancer Society and the Dutch Ministry of Health, Welfare and Sport.
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Contributions
Z.M., R.V., D.R.C., Y.Z., B.R., B.C., H.Y., J.O., J.X., T.N.S., X.C., E.P. and W.S. designed, performed, analyzed and/or interpreted experiments. Z.M. and S.K. analyzed sequencing data. J.B.A.G.H. and D.H. supplied patient tumor material. T.W. and L.R. supplied patient tumor material and isolated and cultured patient TIL lines. J.W. helped conceptualize the methodology. E.P., X.C. and W.S. wrote the manuscript. All authors reviewed the manuscript.
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Competing interests
D.R.C., Y.Z., B.C., S.K., H.Y., J.O., J.X. and E.P. are employees and stock option holders of RootPath, Inc. or its affiliates. X.C. is a director and shareholder of RootPath, Inc. or its affiliates. J.W. is a paid consultant for RootPath Genomics, Bristol Myers Squibb (Relatlimab Advisory Council) and Hanmi Pharmaceutical and is a founder and equity holder of and a consultant to Remunix, Inc. J.B.A.G.H. is an advisor to Achilles Therapeutics, BioNTech, Instil Bio, Neogene Therapeutics, PokeAcell, Scenic Biotech, T-Knife and Third Rock Ventures; is a recipient of research grant support from BioNTech; and is a stock option holder of Neogene Therapeutics. T.N.S. serves as an advisor for Allogene Therapeutics, Merus, Neogene Therapeutics and Scenic Biotech and is a stockholder in Allogene Therapeutics, Cell Control, Celsius, Merus and Scenic Biotech. T.N.S. is also a venture partner at Third Rock Ventures, all outside the submitted work. W.S. is an advisor to BD Biosciences and Lumicks. The TCR library synthesis method is described in patent application WO2020206238A2, assigned to a subsidiary of RootPath, Inc. (X.C. and E.P.). The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Pooled TCR library subcloning and accuracy.
(a, b) Schemes to produce ssDNA CDR3Jα pools (a) and ssDNA CDR3Jβ pools (b). Squares indicate 5′ phosphothioate modification. See Supplementary Table 29 for primer sequences. (c) Complete VCV pools can be PCR-amplified prior to subcloning using the common forward (CF) and reverse (CR) primers. Individual TCRs may be selectively amplified from VCV pools using the common forward primers (CF) and reverse primers targeting their respective Zip barcode sequences. (d) For the proof-of-concept TCR library (553 TCRs), the frequencies of individual CDR3Jα and CDR3Jβ sequences within the commercially synthesized CDR3Jα and CDR3Jβ ssDNA oligo pools and the assembled CDR3Jα-CDR3Jβ product were assessed by deep sequencing. The expected frequency of each CDR3Jα-CDR3Jβ pair was derived by multiplication of the observed frequencies of its respective CDR3Jα and CDR3Jβ sequences in the original, unassembled oligo pools. (e) Relation between Zip and CDR3Jα/CDR3Jβ oligo characteristics and the frequencies of resulting CDR3Jα-CDR3Jβ pairs after hybridization. (f) Relation between Zip and CDR3Jα/CDR3Jβ oligo characteristics and the accuracy of CDR3Jα-CDR3Jβ pairing (‘α-β pairing accuracy’).
Extended Data Fig. 2 ConnA and ConnB orthogonality for murine TRAV and TRBV genes and acquisition accuracies.
(a) Computer-predicted orthogonality of natural (left) and codon-diversified (right) FR3 connector sequences (ConnA) for murine TRAV. (b) Computer-predicted orthogonality of natural (left) and codon-diversified (right) FR3 connector sequences (ConnB) for murine TRBV. (c, d) Acquisition accuracies for TRAV (c) and TRBV (d) genes used in the proof-of-concept TCR library. The average acquisition accuracy for each TRAV/TRBV gene is shown above the bars.
Extended Data Fig. 3 Gating strategies.
(a) Gating strategy for the isolation of CD4+ and/or CD8+ T cells from patient tumor material, for the purpose of identifying patient-derived TCRs by single-cell TCR sequencing (relating to the patient TCR libraries used in Figs. 2 to 4). (b) Gating strategy for the isolation of activated reporter T cells in TCR library screens (relating to Figs. 2b–d, 3a,d and 4a, and Extended Data Figs. 7a–c, and 9b,d. (c) Gating strategy for T cell activation assays (relating to Figs. 3c,e,f and 4b,c, and Extended Data Figs. 7d–f and 9a. CD137 served as activation marker when using primary T cells, and CD69 served as activation marker when using Jurkat cells. (d) Gating strategy for T cell cytotoxicity assays (relating to Figs. 3g and 4d).
Extended Data Fig. 4 Quality control of the NKIRTIL063 TCR library.
(a) Schematic overview of the custom ‘2-reads-on-1-strand’ sequencing method. (b) Overall assembly accuracies and frequencies of TCRs in the fully assembled NKIRTIL063 VCV library (n = 935 TCRs). Dots represent individual TCRs. Box plots depict the median, the interquartile range and whiskers extending to minimal and maximal values. (c) Pie chart depicting the overall fraction of NKIRTIL063 VCV molecules with and without sequence errors (taking both assembly accuracy and sequence mutations into account). (d) The fraction of NKIRTIL063 library TCRs successfully expressed at the cell surface of library-transduced cells was determined by isolation of mouse TCRβ+ Jurkat cells by flow cytometry, followed by deep sequencing. NKIRTIL063 library-expressing Jurkat cells that were not subjected to cell sorting were used as reference. Internal control TCRs DMF4 and DMF5 are highlighted for reference.
Extended Data Fig. 5 Quality control of the OVC190 TCR libraries.
(a) Overall assembly accuracies and frequencies of TCRs in the fully assembled OVC190 CD4+ TIL-derived VCV libraries (combined n = 1,341 TCRs). Selected TCRs were encoded in replicate, resulting in a total of 2,899 unique sequences that were assembled in two separate reactions (SP1 and SP2). Dots represent individual TCRs. Box plots depict the median, the interquartile range and whiskers extending to minimal and maximal values. (b) Overall assembly accuracies and frequencies of TCRs in the fully assembled OVC190 CD8+ TIL-derived VCV library (n = 274 TCRs). (c) Pie chart depicting the overall fraction of OVC190 CD4+ and CD8+ TIL-derived VCV molecules with and without sequence errors (taking both assembly accuracy and sequence mutations into account).
Extended Data Fig. 6 Quality control of the CV19 TCR library.
(a) Overall assembly accuracies and frequencies of TCRs in the fully assembled CV19 TIL-derived VCV libraries (combined n = 1,501 TCRs). Sequences were assembled in two separate reactions (SP1 and SP2). Dots represent individual TCRs. Box plots depict the median, the interquartile range and whiskers extending to minimal and maximal values. (b) Pie chart depicting the overall fraction of CV19 VCV molecules with and without sequence errors (taking both assembly accuracy and sequence mutations into account).
Extended Data Fig. 7 Functional screening of intratumoral TCR repertoire of patient CV19.
(a) The SiHa cervical cancer cell line was engineered to express the entire MHC class I haplotype of patient CV19 (A*24:02, A*33:03, B*38:15, B*55:02, C*03:02 and C*12:03). The CV19 TCR library (n = 1,501 TCRs) was subsequently expressed in donor T cells and screened against either the unmodified or MHC-modified SiHa line. Fold change represents the relative abundance of TCRs after incubating T cells with unmodified or MHC-modified SiHa cells. TCRs selected for validation are highlighted in red. (b, c) To assess TIL reactivity against HPV-derived antigens, the patient TCR library was screened against K562 cells that were modified to express the patient’s MHC class I alleles as well as the full ORF of either HPV E6 (b) or E7 (c) oncoproteins. Data are depicted as in (a). Two of four selected TCRs (2495 and 362) responded to E7-expressing K562 cells, while no TCRs responded against E6-expressing cells. (d) The reactivity of selected TCRs was validated by amplifying TCRs from the CV19 VCV pool, followed by expression in donor T cells and co-incubation with the indicated cell lines. T cell activation was assessed by measuring CD137 expression using flow cytometry. (e, f) MHC restriction of selected TCRs was assessed by expressing individual patient MHC alleles in SiHa cells and incubating resulting cells with donor T cells engineered to express selected TCRs. T cell activation was assessed by measuring CD137 surface expression. (g) SiHa-reactive TCRs 1007 and 3645, but not E7-specific TCRs 362 and 2495, recognize autologous patient tumor cells. TCR-modified donor T cells were incubated with unmodified, HLA-B*55:02-modified or HLA-B*38:15-modified SiHa cells, or autologous dissociated tumor tissue at the indicated effector to target ratios. Activation of TCR-engineered T cells in measured by IFNγ ELISpot.
Extended Data Fig. 8 Sensitive TCR discovery across variable TCR frequencies and sequence fidelities.
(a) Projection of all NKIRTIL063 screen hit TCRs with confirmed (blue, n = 44) and unconfirmed (red, n = 36) reactivity (as reported in Fig. 3) onto the quality control data of the NKIRTIL063 TCR library (see Extended Data Fig. 4). (b, c) Comparison between the frequencies (b) and assembly accuracies (c) of the overall NKIRTIL063 TCR library, TCRs with confirmed reactivity and TCRs with unconfirmed reactivity. Box plots depict the median, the interquartile range and whiskers extending to minimal and maximal values. P values were determined using the Kruskal-Wallis test.
Extended Data Fig. 9 Neoantigen specificities of OVC190 TCRs.
(a) TCR-modified CD4+ Jurkat cells were incubated with patient B cells expressing either the mutant or wildtype sequence of the individual minigenes of TMG 5. T cell activation was determined by measuring CD69 expression on T cells by flow cytometry. (b) CD8+ Jurkat cells were transduced with the CD8+ TIL-derived TCR library of patient OVC190 (n = 274 unique TCRs). Patient immortalized B cells were transduced with TMGs encoding all expressed non-synonymous mutations (n = 61) of the patient’s tumor, combined in pools and used to screen the OVC190 TCR library. Dots represent individual TCRs. Fold change represents the relative abundance of TCRs in cultures with the indicated TMG pools. (c) Flow cytometry analysis of MHC class I (top panels) and MHC class II (bottom panels) expression on CD45+ and CD45- cells within the OVC190 tumor. Fluorescence minus one (FMO) stains with antibody panels that lacked either the panMHC-I or panMHC-II antibody served as negative control. (d) CD8+ Jurkat cells expressing the OVC190 CD8+ TIL-derived TCR library were screened against single cell suspension of OVC190 tumor. Screening the TCR library against tumor cells in the presence of MHC class I blocking antibody (clone W6-32) served as negative control. Data are depicted as in (b).
Extended Data Fig. 10 Patient HC25 tumor-specific TCR identification.
(a) Patient HC25 PD-1+ TIL were sorted by flow cytometry, and subjected to paired single cell RNA and TCR sequencing. NeoTCR4 and NeoTCR8 transcriptional signatures were derived for CD4+ and CD8+ clonotypes, respectively, and clonotypes with the 382 highest scores were gene-synthesized. Individual TCRs were expressed in donor T cells and reactivity to autologous dissociated tumor tissue was assessed by IFNγ ELISpot. Wells with responding TCRs are marked by red asterisks. Red boxes indicate HC25 TCR-independent experimental controls. (b) Selective reactivity of hit TCRs to patient tumor cells, but not non-malignant cells, was validated by incubating TCR-engineered donor T cells with either medium, patient HC25 activated T blasts, or patient HC25 dissociated tumor tissue. T cell activation was assessed by measuring CD137 expression using flow cytometry. Asterisks indicate TCRs with selective tumor-reactivity.
Supplementary information
Supplementary Information
Supplementary Figs. 1–3.
Supplementary Tables
Supplementary Tables 1–29.
POC TCRsZip sequences. POC a-b pairing. POC a-b uniformity. ConnA sequences. ConnB sequences. POC Va-CDR3Ja. POC Vb-CDR3Jb. NKIRTIL063 Ref. NKIRTIL063 QC. OVC190CD4 SP1 Ref. OVC190CD4 SP2 Ref. OVC190CD4 SP1 QC. OVC190CD4 SP2 QC. OVC190CD8 Ref. OVC190CD8 QC. CV19 SP1 Ref. CV19 SP2 Ref. CV19 SP1 QC. CV19 SP2 QC. HC25 Ref. POC Oligos. NKIRTIL063 Oligos. OVC190CD4-SP1 Oligos. OVC190CD4-SP2 Oligos. OVC190CD8 Oligos. CV19-SP1 Oligos. CV19-SP2 Oligos. Other sequences.
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Moravec, Z., Zhao, Y., Voogd, R. et al. Discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02210-6
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DOI: https://doi.org/10.1038/s41587-024-02210-6
