Abstract
Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1,2,3,4,5,6,7,8,9,10,11,12,13,14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15,16,17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen–HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR–Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.
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Data availability
Raw sequencing data derived from cell line and normal PBMC control samples collected under UCLA Institutional Review Board approvals 11-003254 have been deposited to dbGAP under accession number phs003153.v1.p1.
Code availability
The software used to analyse the data include: WA-MEM, Sentieon (v.201911.01), VarScan2 (v.2.3.9 and v.2.4.2), MuTect (v.1.1.7 and v.3.6), HISAT2 (v.2.0.4), STAR, RSEM, StringTie (v.1.2.2), Optitype (v.1.3.1), netMHCpan (v.3.4 and v.4.1), CWL (v.1.1), Strelka (v.2.9.9), Pindel (v.0.2.4b8), pVACtools (v.1.1.4; supported by NetMHCpan (v.4.0), NetMHC (v.4.0), NetMHCcons (v.1.1), PickPocket (v.1.1), SMM (v.1.0), SMMPMBEC (v.1.0), MHCflurry (v.1.2.2) and MHCnuggets (v.2.2)), Kallisto and MiXCR (v.2.1.3).
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Acknowledgements
We thank B. Berent-Maoz and J. Pang for help in sample collections; J. M. Chen and J. Trent for administrative and logistics support; and Y. Qi, Y. Murtanu, L. Guo, M. Dhar, E. Huang and M. Wallace for their help in contributing suggestions and processing the samples. This study was funded in part by PACT Pharma, the Parker Institute for Cancer Immunotherapy (PICI) (to A.R. and to C.P.-S.), the Melanoma Research Alliance—Young Investigator Award (to C.P.-S.), NIH grants R35 CA197633 and P01 CA244118 (to A.R.), the Ressler Family Fund and contributions from K. and D. Schultz, T. and D. Jones, and T. Stutz (to A.R.). C.P.-S. is a Parker Senior Fellow supported by PICI. G.A.-R. was supported by the Isabel and Harvey Kibel Fellowship award and the Alan Ghitis Fellowship Award for Melanoma Research, and currently by a Parker Scholar award; and J.D.S. by the UCLA Tumour Immunology Training Grant (USHHS Ruth L. Kirschstein Institutional National Research Service Award T32 CA009120). Flow Cytometry was performed at the UCLA Jonsson Comprehensive Cancer Center (JCCC) Flow Cytometry Shared Resource that is supported by the National Institutes of Health award P30CA016042 and by the JCCC and the David Geffen School of Medicine at UCLA. Cell sorting was performed at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles Flow Cytometry Core Resource. Sequencing studies were conducted at the UCLA Technology Center for Genomics & Bioinformatics.
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Authors and Affiliations
Contributions
C.P.-S., A.F., S.J.M. and A.R. designed the study and provided overall guidance. C.P.-S. B.S., S.P., Z.P., K.J., O.D., C.L.W., A.F., S.J.M. and A.R. wrote the first version of the manuscript. All of the authors contributed to the final manuscript. C.P.-S., B.S., S.P., Z.P., K.J., O.D., C.L.W., A.F., S.J.M. and A.R. analysed the results. G.C., D.J.W., B.C. and A.R. contributed to patient treatment and care. C.P.-S., S.J., J.D.S., I.P.-G., A.V.-C., I.B.-C., G.C. and A.R. contributed to patient sample collection and processing. Z.P., H.X., Y.M., S.Z., E.M., J. Hundal., O.L.G. and M.G. contributed to bioinformatics analysis. M.Y., C.S., K.H., O.D. and M.T.B. performed the protein library synthesis. S.P., C.L.W., M.T.B., D.A., B.B.Q., B.Y., D.N., S. Said., K.S., J.G., J. Hoover., X.R.B., R.T., C.M., P.P., S.N., J.R.H., A.F. and S.J.M. contributed to the design and/or performance of the neoantigen-specific isolation experiments. K.J., W.L., D.N., S.C., M.J.P., R.M., S.P.F., T.H., A.F. and S.J.M. contributed to the design and/or performance of the gene-editing experiments. C.P.-S., B.S., B.P., A.C., M.T., S.J., J.D.S., A.F., S.J.M. and A.R. contributed to the design and/or performance of the T cell functional studies. G.A.-R., D.E.S. and A.G. provided guidance, samples and materials throughout the different stages of these studies.
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Competing interests
B.S., S.P., C.L.W., Z.P., B.P., A.C., D.A., B.B.Q., B.Y., K.J., O.D., D.N., K.S., J.G., J. Hoover., S. Said., W.L., C.S., K.H., Y.M., S.C., M.J.P., M.T., R.T., C.M., P.P., S.N., S.P.F., T.H., M.Y., S. Sandoval., R.M., X.R.B., M.T.B., A.F. and S.J.M. are current or former employees of PACT Pharma, and hold stock in the company. S.P., M.T.B., J.R.H. and A.R. are co-founders of PACT Pharma, and hold founder stock. J.R.H. and A.R. are members of the scientific advisory board of PACT Pharma. J.R.H. and A.R. are members of the board of directors of PACT Pharma. A.R. has received honoraria from consulting with Amgen, Bristol-Myers Squibb, Chugai, Dynavax, Genentech, Merck, Nektar, Novartis, Roche and Sanofi; is or has been a member of the scientific advisory board and holds stock in Advaxis, Arcus Biosciences, Bioncotech Therapeutics, Compugen, CytomX, Five Prime, RAPT, ImaginAb, Isoplexis, Kite-Gilead, Lutris Pharma, Merus, Rgenix and Tango Therapeutics. C.P.-S. and A.R. are listed as inventors on and receive licensing revenue from a patent application covering the use of non-viral gene editing of T cells that was licensed by The Regents of the University of California to Arsenal Bio (South San Francisco; WO2019084552A1, application filed by The Regents Of The University Of California); the methods and technology described therein were not used in the experiments performed herein. A.G. reports receiving funding from Novartis, VCN Biosciences and Merck KGaA, has received speaker honoraria from Roche, and has consulted for Achilles Therapeutics, Neon Therapeutics, PACT Pharma and Oxford Immunotherapy. Patent applications have been filed on aspects of the described work by PACT Pharma, entitled ‘Peptide-MHC comPACTs’, ‘Compositions and Methods For Identification of Antigen Specific T Cells’ and ‘Primary Cell Gene Editing’. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Neoantigen-specific T-cell isolation and TCR clonotype identification. Summary of key parameters from the longitudinal landscape analysis of neoantigen-specific T cells in patients with and without response to therapy.
a, Total number of non-synonymous mutations. b, Total number of mutations screened. c, Total number of predicted neoantigen–HLA complexes screened. c, Total number of mutations targeted. e, Total number of neoantigen-specific TCR clonotypes isolated. f, Ratio of the number of neoantigen specific TCR clonotypes isolated per mutation targeted in each patient. Mean ± SD and individual values are plotted. (n) indicates the number of different patients, n = 7 for responders and n = 4 for non-responders. * p < 0.05, two-tailed unpaired t test, using the Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. p = 0.0190 for TCR clonotypes and p = 0.0434 for TCR clonotype/mutation targeted.
Extended Data Fig. 2 Neoantigen-specific T-cell isolation from PBMCs in patients with response to anti-PD-1 therapy.
a, Landscape analysis of the neoantigen-specific T cells over time in patient 3. Bottom panel shows mRNA expression and predicted HLA binding affinity of the putative neoantigens screened. Neoantigens targeted by T cells are highlighted in different colours. The same colour code is used in the top panels to show the neoantigen specificity of the isolated T cells. The top panels show the evolution over time of the neoantigen-specific T cells in PBMCs. Each box represents one isolated T cell, each cross is equivalent to ten isolated T cells, and each circle is equivalent to 100 isolated T cells. Each colour represents a different neoantigen-specific T-cell clonotype. The TCR ID is also plotted. The number of isolated T cells is normalized to 100,000 CD8+ T cells using a round up method to plot the data. The mutated gene name, the point mutation, the sequence of the neoantigen, and the HLA are indicated on top of the figure. The T cell clonotypes shown have not been validated by expression in healthy donor T cells and binding to neoantigen–HLA complexes. b, Same as a for patient 4. c, Same as a for patient 5. d, Same as a for patient 7.
Extended Data Fig. 3 Captured neoTCR specific neoantigen–HLA binding validation.
After capture of the neoantigen-specific T cells, the cognate TCR is sequenced, and the sequence used to gene edit healthy donor T cells replacing the endogenous TCR by the neoTCR. The neoTCR specificity and stability are validated by multimer staining of the gene-edited T cells. Only validated TCRs are shown. a-f. Multimer staining of the gene edited T-cell products gated on live cells from patient 1 or Live/CD45+ cells from patients 2, 6, 9, 10, 11 (n = 1 for patient 1, n = 2 for patient 6, 9 and 11, and n = 3 for patients 2 and 10). Mean ± SD and individual values are plotted. (n) indicates the number of technical replicates.
Extended Data Fig. 4 Cytotoxicity induced by neoantigen-specific TCRs from patient 1 upon co-culture with the autologous cell line.
Healthy donor T cells genetically engineered to express the captured neoTCRs from patient 1 were co-cultured with the autologous (M495) or a mismatched cell line (M202). a-b, Specific target-cell killing by neoTCR gene-edited T cells of the autologous cell line and the mismatched control (P:T ratio 5:1, n = 4)). Melanoma cell lines were pre-treated with media with IFNγ 24 h prior co-culture with T cells. The plots are divided between TCRs without killing activity (a) and TCRs with intermediate killing (b). * p < 0.05 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparison (n) indicates the number of biological replicates. Exact p-values provided in Supplementary Information. Mean and individual values are shown. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Extended Data Fig. 5 Titration of cytokine secretion by neoantigen-specific TCRs from patient 1 upon binding to specific neoantigen–HLA multimer.
Healthy donor T cells genetically engineered to express the captured neoTCRs from patient 1 were incubated with increasing amounts of plate-bound neoantigen–HLA multimer specific for each TCR and, 24 h after incubation, the IFNγ, IL-2, and TNF secreted were measured. a, NeoTCRs targeting UBE2J1 mutation presented by HLA-A*24:02. b, NeoTCRs targeting NUP188 mutation presented by HLA-A*24:02. c, NeoTCRs targeting WDR1 mutation presented by HLA-A*03:01. d, NeoTCRs targeting NRP1 mutation presented by HLA-A*03:01. e, NeoTCRs targeting TRAPPC10 mutation presented by HLA-C*04:01. f, NeoTCRs targeting IFNLR1 mutation presented by HLA-A*03:01. g, NeoTCRs targeting PLA2G4A mutation presented by HLA-A*03:01. h, NeoTCRs targeting SLC6A3 mutation presented by HLA-A*03:01. i, NeoTCRs targeting GSDMB mutation presented by HLA-C*12:02. j, Summary EC50 calculated for each TCR and each cytokine. (n = 2), (n) indicates the number of biological replicates. Mean and individual values are shown. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Extended Data Fig. 6 Activation, cytotoxicity, cytokine secretion, and proliferation induced by neoantigen-specific TCRs from patient 2 upon co-culture with the autologous cell line.
Healthy donor T cells genetically engineered to express the captured neoTCRs from patient 2 were co-cultured with the autologous (M489) or a mismatched cell line (M202). a, 4-1BB, OX-40, and CD107a upregulation in the CD8+ neoTCR+ T cells after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). b, percentage of tumour growth inhibition in M489 autologous cell line compared to the cell growth in media alone at 24, 48, 72 and 96 h (n = 4). c, Cytokine release at 24 h after co-culture (n = 3). d, Proliferation of CD8+ neoTCR+ T cells measured by Ki67 mean fluorescence intensity upon 24, 48 and 72 h co-culture with autologous melanoma cell line (M489, top panel) or a mismatched cell line (M202, bottom panel) (n = 3). * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure a, b and c. * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs M202, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure d. Exact p-values provided in Supplementary Information. (n) indicates the number of biological replicates. Mean ± SD and individual values are shown. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Extended Data Fig. 7 Function of the CD8-independent TCRs in gene-edited CD4+ T cells.
a,c,e,g,i. Percentage of CD8+ and CD4+ in the neoTCR+ T cell population. n = 2 for patients 6, 9 and 11, and n = 3 for patients 2 and 10). CD8-dependent TCRs, described as TCRs that require CD8 co-receptor engagement to bind to the MHC-peptide, are marked with a red arrow. b,d,f,h, Percentage of OX-40+ cells in the NeoTCR+CD4+ T cells. (n = 3) *p < 0.05, **p < 0.005, ***p < 0.0005,****p < 0.0001 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparison in figures b, d, and h. The same test without adjustment for multiple comparisons was used in figure f. Exact p-values provided in Supplementary Information. (n) indicates the number of biological replicates. Mean ± SD are shown. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Extended Data Fig. 8 Activation, cytotoxicity, cytokine secretion, and proliferation induced by neoantigen-specific TCRs from patient 6 upon co-culture with the autologous cell line.
Healthy donor T cells genetically engineered to express the captured neoTCRs from patient 6 were co-cultured with the autologous (M490) or a mismatched cell line (M202). a, 4-1BB and OX-40 upregulation in the CD8+ neoTCR+ T cells after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). b, Specific target-cell killing in the autologous cell line (top panel) or a mismatched cell line (bottom panel), (P:T ratio 10:1, n = 4). c, Cytokine release at 24 h after co-culture (n = 3). Melanoma cell lines were pre-treated with IFNγ for 24 h before co-culture with T cells. d, Proliferation of CD8+ neoTCR+ T cells measured by Ki67 mean fluorescence intensity upon 24, 48 and 72 h co-culture with autologous melanoma cell line (M490, top panel) or a mismatched cell line (M202, bottom panel). Melanoma cell lines were pre-treated with IFNγ 24 h prior co-culture with T cells (n = 3). *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure a, b and c. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001 vs M202, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure d. (n) indicates the number of biological replicates. Exact p-values provided in Supplementary Information. Mean ± SD and individual values are shown in a, c and d. Mean and individual values are shown in b. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Extended Data Fig. 9 Neoantigen-specific T-cell isolation from PBMCs in patients without response to anti-PD-1 therapy.
a, Landscape analysis of the neoantigen-specific T cells over time in patient 8. Bottom panel shows mRNA expression and predicted HLA binding affinity of the predicted neoantigens screened. Neoantigens targeted by T cells are highlighted in different colours. The same colour code is used in the top panels to show the neoantigen specificity of the isolated T cells. The top panels show the evolution over time of the neoantigen-specific T cells in PBMCs. Each box represents one isolated T cell, each cross is equivalent to ten isolated T cells, and each circle is equivalent to 100 isolated T cells. Each colour represents a different neoantigen-specific T-cell clonotype. The number of isolated T cells is normalized to 100,000 CD8+ T cells using a round up method to plot the data. The mutated gene name, the point mutation, the sequence of the neoantigen, and the HLA are indicated on top of the figure. The T cell clonotypes shown have not been validated by expression in healthy donor T cells and binding to neoantigen–HLA complexes.
Extended Data Fig. 10 Activation, cytokine secretion, and proliferation induced by neoantigen-specific TCRs from patients without response to anti-PD-1 upon co-culture with the autologous cell lines.
Healthy donor T cells genetically engineered to express the captured neoTCRs from patient9 (a), 10 (b-d) and 11 (e-f) were co-cultured with the autologous (M488, M485 and M486 respectively) or a mismatched cell line (M202). a, 4-1BB upregulation in the CD8+ neoTCR+ T cells from patient 9 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). b, 4-1BB, OX-40, and CD107a upregulation in CD8+ neoTCR+ T cells from patient 10 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). c, Cytokine release at 24 h after co-culture (n = 3). d, Proliferation of CD8+ neoTCR+ T cells from patient 10 measured by Ki67 mean fluorescence intensity upon 24, 48 and 72 h co-culture with autologous melanoma cell line (M485, top panel) or a mismatched cell line (M202, bottom panel) (n = 3). e, 4-1BB and OX-40 upregulation in CD8+ neoTCR+ T cells from patient 11 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). f, Cytokine release at 24 h after co-culture (n = 3). * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure a, b, c, e, and f. * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs M202, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure d. Exact p-values provided in Supplementary Information. (n) indicates the number of biological replicates. Mean ± SD and individual values are shown. All T-cell products contain CD8+ and CD4+ gene-edited T cells.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2, Supplementary Tables 1 and 2 (Supplementary Data) and statistical data tables (P values).
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Puig-Saus, C., Sennino, B., Peng, S. et al. Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy. Nature 615, 697–704 (2023). https://doi.org/10.1038/s41586-023-05787-1
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DOI: https://doi.org/10.1038/s41586-023-05787-1
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