An immunogenic personal neoantigen vaccine for patients with melanoma

a, Numbers of mutations and predicted epitopes per patient tumour. Red solid lines, patients with vaccines generated and vaccinations completed (n = 6); red dashed lines, vaccines generated but vaccinations not initiated (n = 2); grey, insufficient mutation number for vaccine generation (n = 2). b, Expression (measured and normalized as transcripts per million base pairs (TPM) from the RNA-seq data) of known melanoma-associated genes in the tumour specimens for all ten patients entered into the trial (circles) compared with skin tissue (blue box, (GTEx data)) and melanoma from TCGA (red box; see Methods for analysis). The upper, middle, and lower hinge of the box are 75%, 50%, and 25% quantiles. All points above or below whiskers are the outliers. c, The overall mutational landscape of the eight patient melanoma samples for which immunizing peptides were generated (top, number of mutations per megabase; bottom, distribution of nucleotide changes) and the presence of mutations in genes previously identified as recurrent in melanoma TCGA samples (n = 290) by the MutSig2CV algorithm (middle, doi:10.7908/C1J67GCG; genes ordered on the basis of significance of recurrence reported per TCGA, Supplementary Information 2). d, Patient (Pt.) 2. Left: positron emission tomography/computed tomography (PET/CT) scans obtained 8 weeks after the last booster vaccine demonstrate new intensely fludeoxyglucose F 18 (FDG)-avid right hilar lymphadenopathy measuring ~2.1 cm (maximal standardized uptake value 20.3 (yellow arrow)). Right: PET/CT obtained 12 weeks later, after four doses of pembrolizumab at 2 mg per kg (body weight) given every 3 weeks, revealing complete interval resolution of the right hilar lymphadenopathy consistent with a complete response. Patient 6. Left panels: CT scans of the chest obtained 1 week after the last booster vaccine demonstrate multiple soft-tissue nodules along the left lateral and posterolateral chest wall (left upper panel, yellow arrow indicates a 2.1 cm × 1.8 cm soft-tissue nodule as an example) and a left lower lobe pulmonary nodule (left lower panel, yellow arrow). Right panels: CT scans obtained 16.5 weeks later, after four doses of pembrolizumab at 2 mg per kg (body weight) given every 3 weeks, revealing complete interval resolution of all lesions consistent with a complete response. On the upper panels, note that the liver is partly visualized on the pre-treatment scan (left upper panel) as a result of expirational state and not visualized on the post-treatment scan (right upper panel) as a result of inspirational state. The cross sections visualizing the soft-tissue metastases in the left chest wall versus their resolution after treatment correspond.

Frequencies of CD4⁺ T cells secreting cytokines in response to pools of 15- to 16-mer ASP, as measured by ICS after stimulation of PBMCs ex vivo with ASP pools. a, IFN-γ-producing CD4⁺ T cells detected by flow cytometry before vaccination and at week 16 after vaccination for all six vaccinated patients. Fluorescence-activated cell sorting (FACS) plots were pre-gated on CD4⁺ T cells. Red values, frequencies of ASP-pool-reactive IFN-γ⁺ cells as a proportion of all CD4⁺ T cells. b, Frequencies of IFN-γ-, TNF-α-, and IL-2-producing cells within CD4⁺ T cells in response to ASP pools tested in samples collected before vaccination (blue) and at week 16 after vaccination (red). c, Pie charts of the total CD4⁺ T-cell responses positive for one, two, or three cytokines. Bar graphs are shown of absolute frequencies of ASP-pool-reactive CD4⁺ T cells producing one, two, or three cytokines. d, Median percentage cytokine production across six patients by CD4⁺ T cells ex vivo against ASP peptide pools.

Frequencies of CD8⁺ T cells secreting cytokines in response to pools of 9- to 10-mer predicted class I EPT after a single round of pre-stimulation. PBMCs were cultured with EPT pools for 10–21 days followed by ICS. a, IFN-γ-producing CD8⁺ T cells detected by flow cytometry pre-vaccination and at week 16 after vaccination for all six vaccinated patients. FACS plots were pre-gated on CD8⁺ T cells. Percentages shown in red are frequencies of EPT-pool-reactive IFN-γ⁺ cells as a proportion of all CD8⁺ T cells. b, IFN-γ responses of CD8⁺ T cells against EPT pools after one round of pre-stimulation measured by ICS pre-vaccination and at 16 weeks after vaccination. c, Bar graphs show absolute frequencies of EPT-pool-reactive CD8⁺ T cells producing one, two, or three cytokines. Pie charts summarize the fractions of the total CD8⁺ T-cell responses positive for one, two, or three cytokines. d, Median percentage cytokine production across six patients by CD8⁺ T cells after one round of pre-stimulation against EPT peptide pools.

Deconvolution of T-cell reactivity against individual ASP by ex vivo IFN-γ ELISPOT using the week 16 post-vaccination PBMCs, tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). *Responses were scored positive if >55 spot-forming cells (SFC) were detected and were at least 1.5 s.d. over the DMSO control (>3 s.d. over background for patients 5 and 6). a, Patient 3 deconvolution. Inset: examples of positive IMP/ASP sequences; red, ASP stimulating reactivity; boxed, the mutated amino acids. b, Deconvolution for all other patients. c, IFN-γ ELISPOT response of patient 3 CIT-specific CD8⁺ T-cell line from week 16 (7 × 10⁴ CD8⁺ T cells per well) against mutated (Mut) versus corresponding wild-type peptide across a range of concentrations (10 pg ml⁻¹ to 10 μg ml⁻¹) pulsed on 1 × 10⁴ T-cell-depleted PBMCs (hereafter referred to as APCs) per well.

ASP and EPT covering the IMP are shown for the IMP that induced T-cell responses. T cells from week 16 PBMCs were tested. Red bold and shading: mutated amino acids, absent in those IMP arising from neoORFs. Blue underline: for class I epitopes, predicted epitopes (IC₅₀ < 300 nM) based on NetMHCpan^36,45; for class II epitopes, predicted epitopes <10th percentile based on the Immune Epitope Database and Analysis Resource (IEDB)-recommended consensus approach combining NN-align, SMM-align, and CombLib if allele predictions are available, otherwise NetMHCIIpan (Supplementary Information 6)⁴⁵. Red font, peptides that generated an ex vivo CD4⁺ T-cell response; blue font, peptides that generated a T-cell response after one round of pre-stimulation with peptides, Triangle, ASP- or EPT-specific T cells that also recognized a corresponding mutated minigene; *T-cell responses against minigenes that were blocked by pan anti-HLA-DR or anti-HLA class I blocking antibodies for CD4⁺ or CD8⁺ T cells, respectively.

PBMCs from week 16 after vaccination were pre-stimulated with ASP and EPT for measuring CD4⁺ and CD8⁺ T-cell responses, respectively. a, Detection of IFN-γ responses by ELISPOT of CD4⁺ T cells against individual ASP after one round of pre-stimulation with ASP pools in matched pre- and 16-week post-vaccination samples for patients 1–6 (1 × 10⁴ APCs and 1 × 10⁴ T cells per well), tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). b, Autologous B cells were nucleofected with in vitro translated RNA generated from mutated (Mut) or wild-type minigenes (MG). T-cell lines were cultured with mutated or wild-type minigene nucleofected B cells, mutated minigene nucleofected B cells with anti-HLA class I or anti-HLA-DR antibodies, and non-nucleofected B cells with DMSO in IFN-γ ELISPOT (8 × 10⁴ B cells per well were plated with 5 × 10³ T cells per well and 1 × 10⁴ CD8⁺ T cells per well for CD4⁺ and CD8⁺ T-cell response detection, respectively), tested in duplicate or triplicate wells per each condition (error bars, s.e.m.). c, T-cell lines were cultured with mutated minigene nucleofected B cells or non-nucleofected B cells followed by CD107αβ degranulation assay. Representative FACS plot of patient 3 CD107αβ degranulation assay for VPS16-specific CD8⁺ T-cell lines (top) and other neoantigen-specific CD4⁺ and CD8⁺ T-cell lines (bottom) from week 16 against autologous B cells nucleofected or not with mutated minigenes. FACS plots were pre-gated on CD8⁺ T cells.

a, Flow cytometric staining of autologous melanoma cell lines generated from primary tumour samples with anti-class I and anti-class II or isotype antibodies. b, Dual chromogenic immunohistochemical staining of excised FFPE tumours (see Methods for details). Representative images of positive staining for HLA class I (patients 4 and 2) and HLA class II (patient 6) and negative staining for class II (patient 2). Red, melanoma transcription factor SOX10; brown, HLA class I or class II. c, Summary of immunohistochemical results of five patients with available FFPE tissue. Semi-quantitative scoring was performed for the intensity of positive staining of melanoma cell membranes for class I or II (0, negative; 1, weak; 2, moderate; 3, strong) and for the percentage of positive staining malignant cells (0–100%). A cumulative H score was obtained by multiplying intensity score by the percentage of malignant cells with positive staining.

a, Persistent or new responses to mutated peptides after PD-1 blockade (pembrolizumab) are shown. PBMCs from patients 2 and 6 (before vaccination, week 16 after vaccination, or 11.5 months (patient 2) or 8.5 months (patient 6) after initiation of pembrolizumab therapy) were pre-stimulated for 21 days with ASP or EPT pools, and reactivity against individual ASP and EPT was then tested by IFN-γ ELISPOT. Ten thousand APCs per well were plated with 1 × 10⁴ T-cell lines per well and 1 × 10⁴ CD8⁺ T-cell lines per well for CD4⁺ and CD8⁺ T-cell response detection, respectively, tested in duplicate or triplicate wells per peptide (error bars, s.e.m.). Each line represents the IFN-γ response against a single ASP or EPT tested per immunizing peptide. Number for each gene: total number of ASP or EPT tested per immunizing peptide. b, The rings, from outer to inner, show the following: (1) predicted affinity of peptide for HLA; heat map scaled from darkest (lowest half-maximal inhibitory concentration (IC₅₀) (nM), or highest affinity) to lightest (highest IC₅₀ (nM), or lowest affinity); (2) level of transcript expression of the tumour, scaled from darkest (highest number of transcripts per million reads) mapped to lightest (lowest number of transcripts per million reads); (3) reactivity to peptide-pulsed autologous APCs; (4) reactivity to B cells nucleofected with minigenes; (5) reactivity to cultured autologous melanoma cells. c, d, Ex vivo class II tetramer staining of patient 1 CD4⁺ T cells. Visualization of transcriptomes of single CD4⁺ T cells before vaccination and tetramer-positive CD4⁺ T cells after vaccination by t-distributed stochastic neighbour embedding (t-SNE).