Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial

Abstract

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses^1,2 and can function as bona fide antigens that facilitate tumour rejection³. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma^4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load^1,7 and an immunologically 'cold' tumour microenvironment⁸. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4⁺ and CD8⁺ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Extended Data Fig. 1 |. Mutational landscape of patient GBM tumours.

a, The overall mutational landscape of the GBM tumours from 10 enrolled patients (top, number of mutations per Mb; bottom, distribution of nucleotide changes) and the presence of mutations in genes that have previously been identified as recurrent in GBM TCGA samples (n = 290) by the MutSig2CV algorithm (middle; genes are ordered on the basis of the significance of recurrence reported in the TCGA). b, Expression profiles of the GBM specimens from patients at study entry, according to RNA-seq analysis (Methods). This analysis revealed that tumour samples from the patients showed: EGFR upregulation (consistent with EGFR amplification and polysomy 7), MDM4 upregulation (consistent with p53 pathway dysregulation), TERT upregulation, upregulation of genes associated with glial intermediate filaments (for example, GFAP, VIM and NES), upregulation of markers of GBM stemness (for example, SOX2 and NOTCH2), Rb pathway dysregulation (for example, RB1, CDKN2A and CDKN2B downregulation and CDK4 and CDK6 upregulation), PTEN downregulation (consistent with monosomy 10) and upregulation of IL13RA2 and BIRC5 (which encodes survivin). Tumour RNA-seq data were available for 9 out of 10 study subjects (red dots, vaccinated, n = 7; blue dots, not included in study because of progressive disease, n = 1; black dots, not included in study because of insufficient numbers of binders, n = 1) and were normalized as transcripts per million base pairs (TPM). These data were compared to expression data from normal brain tissue (GTEx data, blue box) and a published cohort of GBMs (TCGA, red box). The upper, middle and lower hinges of the box plot are 75th, 50th and 25th quartiles, the whiskers extend to 1.5× the interquartile range below and above the lower and upper hinge, respectively, and points above or below the whiskers represent outliers. c, Numbers of mutations and predicted epitopes for all study subjects (Supplementary Table 4). Red lines, patients for whom vaccines were generated (n = 8); grey solid line, patient who was removed from the study before vaccine administration owing to progressive disease (n = 1); grey dotted line, patient had an insufficient number of mutations to proceed to vaccine generation (n = 1).

Extended Data Fig. 2 |. Mapping of CD4⁺ and CD8⁺ T cell responses to individual ASP and EPT to the immunizing peptides for patients 7 and 8.

ASP and EPT that cover the immunizing peptides (IMPs) are shown for the immunizing peptide(s) that induced T cell responses. Blue bold, germline amino acid; red bold, mutated amino acid. Blue underline, predicted class I epitopes (IC₅₀ < 500 nM) based on NetMHCpan^,.

Extended Data Fig. 3 |. Neoantigen-specific T cell responses generated by vaccine in patients.

a, Ex vivo IFNγ ELISPOT observed in a patient treated with dexamethasone during vaccine priming (patient 4, left) showed no response at 12 weeks after vaccination, whereas a patient who did not receive dexamethasone (patient 8, right) showed strong response already at 8 weeks after vaccination. b, Deconvolution of post-vaccination CD4⁺ and CD8⁺ immune responses following stimulation to the neoantigen vaccine pools using IFNγ ELISPOT assays. n = 3 biologically independent samples, data are mean ± s.d. c, SLX4^MUT and ARHGAP35^MUT CD8⁺ T cell responses are only detected by ELISPOT assay in samples 16 weeks after vaccination, not pre-vaccination samples after stimulation in vitro for three weeks. d, Representative IFNγ ELISPOT responses from dominant neoantigen-specific T cell lines established from week 8 or week 16 PBMCs of vaccinated patients against minigene-transfected patient B cells, demonstrating epitope processing and presentation; ELISPOT experiments were performed in triplicate wells per time point. e, Analysis of ex vivo T cell responses to neoantigen pools using intracellular cytokine staining followed by flow cytometry. Data are representative of results from three independent experiments. f, IL-2 producing pool A-reactive T cells isolated ex vivo from patient 8 express CD45RO (memory) and PD-1 (activation) markers. Data are representative of results from two independent experiments.

Extended Data Fig. 4 |. Characterization of tumour expression markers and HLA class I and class II in GBM.

a, The GBM tumour cell line from patient 7 that was established from the initial resection is positive for GBM tumour markers by immunohistochemistry. b, The GBM tumour cell line generated from initial resection tissue of patient 7 has low HLA-class I expression that is upregulated by IFNγ treatment and is negative for HLA-class II DR expression. This experiment was repeated four times. c, Immunohistochemistry analysis of HLA-class I and II expression on diagnostic sections of GBM tumours from the eight patients enrolled and vaccinated in the study. This experiment was performed once, with the available resection tissue. d, Neoantigen-specific CD8⁺ T cells generated by vaccination of patient 7 do not recognize the GBM tumour cell line generated from initial resection tissue of patient 7 by TNF ELISPOT assay with or without IFNγ treatment to upregulate HLA-class I expression on tumour cells. Data were generated from one experiment with three replicate wells. Additional direct tumour recognition assays performed with IFNγ ELISPOT demonstrated similar results (data not shown). e, IFNγ secretion by the neoantigen-specific CD4⁺ T cell line generated from PBMC of patient 7 against autologous dendritic cells co-cultured with irradiated autologous GBM. Class II blocking antibody attenuated the IFNγ response. All T cell lines originated from week 16 PBMCs; ELISPOT experiments were performed in triplicate wells per time point.

Extended Data Fig. 5 |. Analysis of tumour morphology, immune infiltration at initial resection and relapse, and TCR clonotypes of patient 8.

a, Haematoxylin and eosin staining of initial and post-vaccination resection samples show the most-prominent changes for patients 4, 7 and 8, including high levels of perivascular lymphocytes, extensive cystic changes and necrosis post-treatment in patient 7, low tumour content and patchy lymphocytic infiltration in patient 8 and sarcomatous morphology with myomatous changes in patient 4. b, No FOXP3⁺ and CD25⁺ CD4⁺ cells were detected in matched initial and relapse tumour sections, evaluated by multiplex immunofluorescence. c, No correlation was observed between the number of predicted neoantigens and the extent of T cell infiltration in the initial resection sample. d, Schema of single-cell TCR analysis of neoantigen-reactive T cells isolated from post-vaccination PBMCs of patient 8 and comparison to bulk TCR sequencing of cDNA from pre- and post-vaccination PBMCs and from initial and relapse fresh-frozen tumour biopsies. e, TCR clonotypes observed in neoantigen-reactive T cell lines generated from PBMCs of patient 8, based on single-cell-targeted TCRαβ sequencing (see Methods). This experiment was performed once, with the available resection tissue.

Extended Data Fig. 6 |. Analysis of tumour-associated T cells from patient 7.

a, Expression levels (log₂(TPM/10 + 1)) of selected marker genes for regulatory, cytotoxic and naive/memory T cell phenotypes; and expression of co-inhibitory receptors and effector cytokines based on scRNA-seq of tumour-associated CD4⁺ and CD8⁺ T cells from the relapse specimen of patient 7 (top). The average levels of these gene sets were used to define the expression scores of these signatures (bottom). b, Expression levels of selected marker genes for all tumour-associated CD3⁺ T cells from patient 7, including those unable to be resolved as CD4 or CD8 based on scRNA-seq. ND, not determined. c, Expression program associated with cytotoxicity in CD8⁺ (x axis) and CD4⁺ (y axis) T cells. For CD8, we compared all CD8⁺ T cells to non-T_reg CD4⁺ T cells. For CD4, we divided the non-T_reg CD4⁺ T cells by their average expression of a predefined cytotoxic signature (PRF1, NKG7, GZMK, GZMA and CST7) into three groups (low, medium and high) and compared the high to the low groups. The expression log₂ ratios of all expressed genes for these two comparisons (CD8 and CD4) are shown. To identify significant differences, we performed a one-sided permutation test, which shuffled the assignments of cells to groups 100,000 times and defined P values based on the number of times the shuffled log₂ ratios are higher (or lower for negative effects) than the observed log₂ ratios. P values were adjusted by Bonferroni correction and significant genes were defined as those with an adjusted P value below 0.05 and a fold change above 2 (Supplementary Table 10). Significant genes are shown as red dots; significant genes that have also been identified previously are shown in blue font. d, Multiplex immunofluorescence demonstrates increase in CD8⁺PD-1⁺ cell infiltration at relapse in patients who did not receive dexamethasone during vaccine priming (red). Infiltrates were determined by enumerating the mean number of CD8⁺PD-1⁺ cells in 20× fields. The number of fields evaluated per sample was: 4 fields for relapse samples from patients 7 and 8; 5 fields for initial and relapse samples from patient 3, relapse samples from patient 5 and initial samples from patient 8; and 6 fields for initial and relapse samples from patient 4, and initial samples from patients 5 and 7. Data are mean ± s.e.m. P values are two-sided and based on model F-tests. e, Enumeration of the SOX2⁺PD-L1⁺ cells in matched initial and relapse tumour sections, evaluated by multiplex immunofluorescence. Infiltrates were determined by enumerating the mean number SOX2⁺PD-L1⁺ cells in 20× fields as in d. Data are mean ± s.e.m.

Extended Data Fig. 7 |. Single-cell TCR sequencing analysis of tumour-associated T cells from patient 7.

a, Single-cell TCR sequencing analyses of tumour-associated T cells, CD4⁺ neoantigen pool-reactive T cells from peripheral blood ex vivo and CD8⁺ neoantigen-reactive T cell lines (stimulated with ARHGAP35^MUT and SLX4^MUT) show enrichment of particular clonotypes. b, Sorting strategy for isolating neoantigen pool-reactive CD4⁺ T cells from peripheral blood ex vivo. c, Six TCRs identified in both tumour-associated and neoantigen-reactive T cells from peripheral blood were successfully cloned and expressed in the reporter cell line, as verified by stabilized CD3 surface expression; this experiment was repeated twice in independent experiments. The CDR3 sequences of these TCRs can be found in Supplementary Table 9. d, The largest CD8⁺ clone detected among the neoantigen-reactive T cell lines established from peripheral blood of patient 7 was experimentally confirmed to be specific for EPT12A, the MHC class I predicted epitope of ARHGAP35^MUT. Two-sample two-sided t-tests with Welch correction were used for the comparisons; n = 4 biologically independent replicates; data are mean ± s.d. e, CD4⁺ ARHGAP35-specific H02 and F10 TCRs (as described in Fig. 4) discriminate between the mutant and wild-type form of the peptide. n = 2 biologically independent samples, each with two technical replicates; data are mean ± s.e.m. f, The ARHGAP35 mutation in the tumour of patient 7 is present in both the initial and relapse tumour specimens, as visualized by the IGV.

Extended Data Fig. 8 |. Comparison of single-cell expression profiles of circulating neoantigen-stimulated CD4⁺ T cells and tumour-associated CD4⁺ T cells isolated from patient 7.

a, Single-cell transcriptome analysis of CD4⁺ tumour-associated T cells (n = 185), freshly isolated at the time of relapse and neoantigen-reactive CD4⁺ cells isolated from post-vaccination (week 16) peripheral blood (n = 104) of patient 7 (excluding CD4 dropouts). Tumour-associated T cells expressed more granzyme A than circulating neoantigen-reactive CD4⁺ cells and showed higher expression of the co-inhibitory molecule TIGIT. b, Significantly altered expression of CCR7, GZMA, LAG3, PD1 and TIGIT was detected between CD4⁺ cells from the blood versus brain by two-sided Wilcoxon rank-sum tests. The expression levels of ARHGAP35^MUT-specific single T cells (clones H02 (red cross) and F10 (green box)), as described in Fig. 4, are marked. c, Vaccination peptide pool-reactive T cells from patients 7 and 8 PBMCs were stained for TIGIT and TIM3, confirming the minimal expression of these markers on neoantigen-reactive IFNγ-producing T cells in the periphery, compared to IFNγ⁻ controls, as suggested by the single-cell transcriptome data in a. Data are representative of results from two independent experiments.

Extended Data Fig. 9 |. Detection of ARHGAP35^MUT-specific T cells in patient 7, week 16.

a, Thawed PBMCs from week 16 collected from patient 7 revealed 60% of the live PBMCs to be CD3⁺ T cells. Data are representative of results from two independent experiments. b, Thawed PBMCs from patient 7 at week 16 were tested ex vivo by ELISPOT, in which 2 × 10⁵ PBMCs were added per well and exposed overnight to 10 μg ml⁻ of peptides covering ARHGAP35^MUT (ASP34 or ASP35 peptides) compared to negative control (OVA peptide). Experiment was performed once in triplicate wells. c, Results of ex vivo ELISPOT (n = 3 biologically independent samples). Together, these results indicate that the frequencies of ASP35- and ASP34-reactive T cells were 39 and 29 (after subtracting background) per 360,000 T cells, respectively. Detection of ASP35-reactive T cells (F10) and ASP34-reactive T cells (H02) in brain at relapse was 1 each among 277 single intracranial T cells (Fig. 4c). The rate of T cells that recognize immunizing neoantigens is highly enriched in the brain compared to the periphery, P = 0.030 for ASP35 and P = 0.023 for ASP34, two-sided Poisson test.

Fig. 1 |. Generation of a personal neoantigen-targeting vaccine for newly diagnosed patients with glioblastoma that had unmethylated MGMT promoters.

a, Somatic mutations were identified by Clinical Laboratory Improvement Amendments (CLIA)-certified whole-exome sequencing of DNA from surgically resected glioblastoma and matched normal cells (PBMCs) and their expression was confirmed by tumour RNA-seq. Immunizing peptides were selected based on HLA class I binding predictions (Methods). Each patient was vaccinated with up to 20 long peptides, administered in non-rotating pools of 3–5 peptides. b, Clinical event timeline for the eight patients who received at least one vaccine dose, from surgery until time of death due to progressive disease. Blue bars, dexamethasone dose and duration. Grey bars, salvage therapy administered following progression. Median progression-free survival (PFS) and overall survival (OS) was 7.6 months (90% confidence interval, 6.2–9.5) and 16.8 months (90% confidence interval, 9.6–21.3), respectively.

Fig. 2 |. Vaccination induces circulating neoantigen-specific T cell responses in patients who did not receive dexamethasone during vaccine priming.

a, Ex vivo IFNγ ELISPOT responses for PBMCs that were stimulated with neoantigen-peptide pools. Data are background-subtracted; n = 3 biologically independent samples, data are mean ± s.e.m.; Wilcoxon signed-rank test, two-sided without adjustment for multiple comparisons, see Methods for statistical analysis; SFC, spot- forming cells. b, IFNγ secretion by neoantigen-reactive T cell lines from patients 7 and 8 shows discrimination between mutated and wild-type peptides, focusing on neoantigens generating CD4⁺ and CD8⁺ T cell responses. n = 3 biological independent samples, data are mean ± s.d.; SHANK2 responses were detected ex vivo. c, Dominant responses of CD4⁺ and CD8⁺ neoantigen-specific T cell lines from patients 7 and 8 are directed against endogenously processed and presented peptides, as shown by reactivity of these cells against autologous B cells that were nucleofected with minigenes (MG) that encoded these neoantigens. n = 3 biologically independent samples, data are mean ± s.d. d, Ex vivo intracellular cytokine staining of PBMCs from patients 7 and 8 post-vaccination after neoepitope stimulation. PBMCs were pre-gated on CD3⁺ and CD4⁺ T cells. Cytokine-producing neoantigen-reactive cells express CD45RO and PD-1, demonstrating an antigen-experienced memory T cell phenotype. SSC, side scatter; All T cell lines originated from week 8 or 16 PBMCs; ELISPOT experiments were performed in triplicate wells per time point.

Fig. 3 |. Increase in T cell infiltration evident in patients with circulating neoantigen-specific T cell responses.

a, Multiplex immunofluorescence staining of five pairs of initial and relapse tumour specimens reveals increased T cell infiltration in patients who did not receive dexamethasone during vaccine priming (red) compared to those who did (blue), with representative sections shown. Sections show SOX2 (magenta) to stain tumour cells, CD4 (yellow) and CD8 (green). Infiltrates were determined by enumerating the mean number of CD4⁺ and CD8⁺ cells in 20× fields. The number of fields evaluated per sample was: 4 fields for initial and relapse samples of patients 3 and 8, initial samples of patient 4 and relapse samples of patient 7; and 5 fields for relapse samples of patient 4, initial and relapse samples of patient 5 and initial samples of patient 7. Data are mean ± s.e.m. P values reported are two-sided and based on model F-tests. See Methods for statistical analysis. b, Each dot represents a single T cell, and cells with identical TCRs are clustered together. Two dominant SHANK2 neoantigen-reactive TCRαβ clones (dark and light blue) were identified in T cell lines that were generated from Week 16 post-vaccination PBMCs from patient 8 using an IFNγ secretion assay. These clones were detected among post-vaccination (relapse) but not pre-vaccination (initial) tumour-associated TCRαβ sequences using bulk RNA-seq. Two dominant SVEP1-reactive TCRαβ (red and orange) clones were observed in post-vaccination PBMCs but not in brain tumour biopsies. c, Enumeration of the numbers of single CD4⁺ and CD8⁺ tumour-associated T cells that expressed effector cytokines in patient 7, analysed using scRNA-seq. d, CD4⁺ and CD8⁺ tumour-associated T cells in patient 7 expressed multiple co-inhibitory receptors based on scRNA-seq.

Fig. 4 |. TCRαβ clonotypes are shared between tumour-associated T cells and neoantigen-reactive T cells in peripheral blood, with select clones demonstrated to be specific for neoantigens targeted by the vaccine.

a, Schema of single-cell transcriptome and TCR analysis of T cells isolated from the relapse tumour specimen, and from PBMCs stimulated in vitro with immunizing neoantigens from patient 7. b, Select TCRαβ clones are shared between tumour-associated T cells and neoantigen-reactive T cells in the periphery. Each dot represents a single T cell, and cells with identical TCRs are clustered together. c, CD4⁺ TCRs H02 and F10, expressed in JurkatΔαβ cells, are specific for ARHGAP35^MUT, a neoantigen targeted by vaccination for patient 7. H02 TCR responds predominantly to ASP34, whereas F10 responds to ASP35. Two-sample two-sided t-tests with Welch correction used for comparison to negative control ovalbumin peptide (OVA); Irr Pool, irrelevant peptide pool; PMA, phorbol 12-myristate 13-acetate; WT, wild- type amino acid. Top left n = 3 biologically independent samples, all others n = 2 biologically independent samples, each with two technical replicates. Representative data from one of three independent experiments are shown; data are mean ± s.d. d, Distribution of expression levels (log₂(transcripts per million/10 + 1)) for select genes in CD4⁺ infiltrating T cells, highlighting the ARHGAP35^MUT-specific T cells, H02 and F10; blue dots, other cells with TCR clones shared between tumour-associated and neoantigen-reactive T cells in peripheral blood.