Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma

Abstract

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

Conflict of interest statement

Competing Financial Interests

Z.H. is a current employee of ElevateBio. J.S. is a current employee of Moderna Therapeutics. E.F.F is an equity holder and consultant for BioNTech, and equity holder and SAB member of BioEntre. N.H. and C.J.W. are equity holders of BioNTech. N.H. is a consultant for Related Sciences. P.A.O. has received research funding from and has advised Neon Therapeutics, Bristol-Meyers Squibb, Merck, CytomX, Pfizer, Novartis, Celldex, Amgen, Array, AstraZeneca/MedImmune, Armo BioSciences and Roche/Genentech. C.J.W. is subject to a conflict of interest management plan for the reported studies because of her former competing financial interests in Neon Therapeutics, which was acquired by BioNTech. Under this plan, C.J.W. may not access identifiable data for human subjects or otherwise participate directly in the Institutional Review Board-approved protocol reported herein. C.J.W.’s contributions to the overall strategy and data analyses occurred on a de-identified basis. Patent applications have been filed on aspects of the described work entitled as follows: ‘Compositions and methods for personalized neoplasia vaccines’ (N.H., E.F.F. and C.J.W.), ‘Methods for identifying tumour specific neo-antigens’ (N.H. and C.J.W.), ‘Formulations for neoplasia vaccines’ (E.F.F.) and ‘Combination therapy for neoantigen vaccine’ (N.H., C.J.W. and E.F.F.). The Dana-Farber Cancer Institute, the lead site of this trial, has a proprietary and financial interest in the personalized neoantigen vaccine. P.B. reports equity in Agenus, Amgen, Breakbio Corp., Johnson & Johnson, Exelixis, and BioNTech. S.J.R. receives research support from Merck, Bristol Myers Squibb, Affimed, and KITE/Gilead, and is on a scientific advisory board (SAB) for Immunitas Therapeutics. S.A.S. reported nonfinancial support from Bristol-Myers Squibb outside the submitted work. S.A.S. previously advised and has received consulting fees from Neon Therapeutics. S.A.S. reported nonfinancial support from Bristol-Myers Squibb, and equity in Agenus Inc., Agios Pharmaceuticals, Breakbio Corp., Bristol-Myers Squibb and Lumos Pharma, outside the submitted work. B.L.P is a founder of Resolute Bio and Amide Technologies; both companies develop protein and peptide therapeutics. E.I.B. consults for Apexigen, Novartis, Partner Therapeutics and receives clinical trial support from Eli Lilly, Novartis, BMS, Genentech and BVD. K.W.W. serves on the scientific advisory board of TCR2 Therapeutics, T-Scan Therapeutics, SQZ Biotech, Nextechinvest and receives sponsored research funding from Novartis. He is a co-founder of Immunitas, a biotech company. These activities are not related to the research reported in this publication. D.B.K. has previously advised Neon Therapeutics and has received consulting fees from Neon Therapeutics. DBK owns equity in Aduro Biotech, Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Bristol Myers Squibb, Celldex Therapeutics, Editas Medicine, Exelixis, Gilead Sciences, IMV, Lexicon Pharmaceuticals, Moderna, Regeneron Pharmaceuticals. BeiGene, a Chine biotech company, supports unrelated research at TIGL. The remaining authors declare no competing interests.

Figures

Extended Data Figure 1.. Genomic and immunofluorescence analysis of pre-vaccination and recurrent tumors.

a. Evolution of tumor clones post-NeoVax treatment. Top panel. Sites of primary and relapse tumors in the five patients. Relapse tissue sites are different from the primary in all patients except Pt. 3. Middle panel. Change in cancer cell fraction (CCF) of tumor clones (depicted by different colors) between the pre- and post-vaccination time points (T0 and T1 respectively). Gene targets of the vaccine are indicated next to their corresponding clones. Bottom panel. Phylogenetic relationship between the different clones evolving from the main clonal population (common ancestor). Known cancer drivers are indicated next to their corresponding clones. b. The ARHGEF15, MECOM, and NLRC4 mutations originally targeted by neoantigen vaccination for Pt. 1 are not found in the recurrent tumor specimen, as visualized by the IGVc. Multiplex immunofluorescence staining for SOX10, CD8, FOXP3, PD-1, and PD-L1. In the left panel, changes in 1) the cell densities of CD8+ and FOXP3+ TILs, 2) the ratio of FOXP3+ to CD8+ TILs, 3) the percentages of CD8+ TILs and FOXP3+ TILs expressing PD-1, and 4) the PD-L1+expression of SOX10+ melanoma cells are portrayed between pre-vaccine versus recurrent tumor samples for 4 patients with available paired tumor specimens. Pts. 3 and 5 (red lines) had late recurrences, while patients 2 and 6 (black lines) had recurrence during vaccinations and 4 weeks after completion of vaccinations, respectively. Data were derived from 5 to 11 fields of view (median 7) per sample, displayed as mean +/− standard error, and compared using two-sided Wilcoxon rank sum tests. d. Representative multiplexed immunofluorescence micrographs (from among 5 to 11 fields of view per sample) from pre-vaccine versus recurrent tumor samples for the 4 patients with paired tumor specimens. Scale bar = 20 μm.

Extended Data Figure 2.. Vaccination induces strong multi-functional CD4+ T cell responses in two additional patients with high-risk melanoma.

a. Exemplary schema of immunizing (IMP), assay (ASP), and epitope (EPT) peptides. Mutated amino acid is shaded. Each predicted epitope peptide is in colored text and its location is underlined below the IMP peptide. b. PBMCs collected pre- and post- vaccination were tested with individual peptides after one round of in vitro stimulation by IFN-γ ELISpot using week 16 post-vaccination PBMCs, tested in triplicate wells per peptide (error bars, s.e.m.). Only results from positive peptides are shown. c. Deconvolution of T cell reactivity against individual ASP after one round of in vitro stimulation by IFN-γ ELISpot using week 16 post-vaccination PBMCs, tested in triplicate wells per peptide (error bars, s.e.m.). *Responses were scored positive if spot-forming cells (SFC) were at least 2.5-fold over the DMSO control. d. Dual chromogenic immunohistochemical staining of FFPE tumor samples from Pts. 11 and 12 (see Methods for details) for HLA class I and HLA class II. Red: SOX10 (melanoma transcription factor); brown: HLA class I or class II. Representative images of 5 to 11 fields of view (median 7) are shown. e. Summary of immunohistochemical results of seven 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.

Extended Data Figure 3.. Mapping of CD4+ and CD8+ T cell responses to individual ASP and EPT to the IMP for Pts. 11 and 12.

ASP covering the IMP are shown for the IMP that induced T cell responses in Pts. 11 and 12. T cells from week 16 PBMCs were tested. Red bold and shading: mutated amino acids, absent in IMP arising from neoORFs. (Supplementary Dataset 4). Blue font: peptides that generated a T cell response after one round of in vitro pre-stimulation with individual peptides.

Extended Data Figure 4.. Neoantigen-specific CD4+ T cells are isolated at serial time points after vaccination using tetramer staining and harbor diverse TCR clonotypes.

a. IFN-γ secretion measured by ELISpot from neoantigen-specific T cells co-cultured with B cells nucleofected with minigenes (MG) encoding wildtype or mutant neoantigens chosen for tetramers, with and without anti-HLA DR antibodies (“block”), tested in duplicate wells/condition for Pt. 1 and triplicate wells/condition for all other Pts (error bars, s.e.m.) (figure from our previous publication). b.Ex vivo HLA class II tetramer staining of Pts. 1, 3 and 5 CD4+ T cells at a series of time points (pre-vaccination, weeks 3-24) following vaccination. Flow plots were pre-gated on CD4+ T cells. c. Schema of representative single-cell TCR and single-cell RNA sequencing analysis of non-specific CD4+ T cells and neoantigen-reactive CD4+ T cells isolated from pre-vaccination PBMCs and post-vaccination PBMCs, respectively, of Pt. 3. d. All TCR clonotypes observed in tetramer-positive T cells generated from PBMCs of Pts. 3, 4 and 5 based on single-cell-targeted TCRαβ sequencing. +: antigen-reactive following mut-ADAMT27 peptide stimulation of selected TCR clonotypes engineered into allogeneic T cells, −: antigen-non reactive.

Extended Data Figure 5.. Neoantigen-specific CD4+ T cells exhibit transcriptional changes through vaccination.

a. Quantification of tetramer-specific or non-tetramer-specific (pre-vaccination) CD4+ T cells isolated at each time point colored by patient. b. Quantification of tetramer-specific or non-tetramer specific (pre-vaccination) CD4+ T cells isolated at each time point colored by cluster. c. Additional selected feature plots of cluster marker genes. d. Quality control metrics for all clusters. e. i) Patient membership by cluster (left); ii) Cluster membership by patient (center); and iii) time point membership by cluster (right). f. Numbers of neoantigen-specific CD4+ T cells in each i) patient by cluster (left); ii) cluster by patient (center); and iii) time point by cluster (right). g. Heatmaps generated from single-cell transcriptome analysis of CD4+ neoantigen-specific and non-neoantigen specific T cells from Pts. 3 (n = 383), 4 (n = 469), and 5 (n = 370) showing selected immunologic genes.

Extended Data Figure 6.. TCR reconstruction and expression in T cells for reactivity screening.

T cell receptors were selected upon TCR sequencing of tetramer positive CD4+ T cells sorted from 3 patients (Pt 2, 3, 6). These TCRs represented clonotypes that were observed in more than one cell (either as replicates at a single timepoint or at more than one timepoint) or as singletons (observed as a single replicate at a single timepoint). The full-length TCRA and TCRB chains were cloned into a lentiviral vector and expressed in donor T cells through lentiviral transduction (Methods). Transduced T cells were co-cultured with patient-derived B cell lines immortalized with EBV virus and pulsed with different doses of mutated or wild-type neoantigen peptides. TCR reactivity was measured through detection of CD137 surface expression on CD4+ TCR transduced lymphocytes by flow cytometry. The values in the graphs report the percentage of CD137 positive cells with subtraction of background, measured upon coculture of T cells with patient-derived B cells pulsed with DMSO (negative control). Bulk data is showing IFN-γ secretion by neoantigen-reactive T-cell lines against mutated and wild-type peptides at several doses (figure from previous publication), tested in triplicate wells/condition (error bars, s.e.m.).

Extended Data Figure 7.. Summary of all CD4+ and CD8+ T cell responses against neoantigen IMPs across all patients.

For each patient, CD4+ responses are shown above and CD8+ responses below. Immunogenic responses are shown in table format and non-immunogenic epitopes are listed below. In each table, from left to right, are shown the following: (1) light blue: ex vivo immune response detected at week 16 of vaccination, (2) dark blue: in vitro immune response detected at week 16 of vaccination, (3) green: in vitro immune response detected after pembrolizumab (only applicable in Pts 2 and 6; otherwise not applicable (gray), (4) dark red: ex vivo immune response detected long-term, (5) orange: in vitro immune response detected long-term. Numbers are IMP numbers. White numbers indicate the number of ASP (CD4+) and EPT (CD8+) peptides stimulating immune responses. Yellow highlight indicates mutations not found in recurrent tumors (applicable in Pt. 1 only). Note: all ex vivo responses were also detectable in vitro; thus, the in vitro responses shown were not detectable ex vivo.

Extended Data Figure 8.. Induction of polyfunctional neoantigen-specific CD4+ T cell responses after vaccination in Pts. 1 and 3.

a. ASP covering the IMP are shown for the IMP that induced ex vivo T cell responses in Pts. 1 and 3 at week 16. T cells from week 16 PBMCs were tested. Red bold and shading: mutated amino acids. Blue underline: 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. Red font, peptides that generated an ex vivo CD4+ T cell response. These data were previously reported. b. Frequencies of Pt. 3 CD4+ T cells from week 16 and 47 months that were secreting cytokines as measured by ICS after stimulation of PBMCs ex vivo with peptides in Panel a. c. Frequency of PD-1 expression among total, non-cytokine producing, and cytokine-producing CD4+ T cells for Pt. 3. d. Representative flow plot and frequencies of Pt. 1 CD4+ T cells from 55 months that were secreting cytokines as measured by ICS after stimulation of PBMCs ex vivo with peptides in Panel a. e. Frequency of PD-1 expression among total, non-cytokine producing, and cytokine-producing CD4+ T cells for Pt. 1. f. Pie charts depict PD-1 expression, T cell phenotypes, and secretion of individual cytokines among the cytokine-producing CD4+ T cells for Pt. 1. Markers were selected to evaluate cytokine secretion (IFN-γ, IL-2, and TNF-α), activation (PD-1), naïve (blue, CD27+/CD45RA+), effector (red [not visible], CD27−/CD45RA+), central memory (purple, CD27+/CD45RA−), and effector memory (orange, CD27−/CD45RA−) T cell phenotypes.

Extended Data Figure 9.. Mapping of individual ASP and EPT directed CD4+ and CD8+ T cell responses to the non-vaccine IMP and TAAs, and tracking of unique TCR clonotypes identified through single-cell and bulk TCR sequencing during and after vaccination.

a. ASP and EPT covering the non-vaccine IMP that induced T cell responses. Blue font: peptides that generated a T cell response after one round of pre-stimulation with peptides. Red highlight: mutated amino acids. EPT covering the TAA are shown for the TAA that induced T cell responses. Blue underline: assay peptides with class II prediction rank of < 10th percentile by NetMHCIIpan. Green underline: assay peptides with prediction rank of < 10th percentile by NeonMHC2. Orange underline: assay peptides with class II prediction rank of <10th percentile by both NetMHCIIpan and NeonMHC2. (Supplementary Datasets 11, 12). b. TCRαβ clonotypes for Pts. 2-6 originally identified through single-cell TCR sequencing that were identifiable at the long-term time points by bulk TCR sequencing are shown. Beige circles indicate time points of detection by single-cell TCR sequencing. Black circles indicate timepoints of detection by bulk TCR sequencing.

Extended Data Figure 10.. Flow cytometry gating strategy for tetramer staining assays and multiparameter intracellular cytokine staining.

a. Representative flow cytometry gating strategy for tetramer staining assays. Gating scheme was used for assays shown in Figure 2a, Figure 4a and Extended Data Figure 4b. b. Representative flow cytometry gating strategy for multiparameter intracellular cytokine staining assay. PD-1 and CD27/CD45RA gating only shown for IFN-γ; similar gating was performed for TNF-α and IL-2. Gating scheme was used for ICS shown in Figure 5d and Extended Data Figure 8b-f.

Figure 1.. Long-term clinical outcomes of neoantigen-vaccine treated melanoma patients and immune responses in two newly vaccinated patients.

a. Clinical course of 8 patients (Pts) who received personalized neoantigen vaccines starting at the time of melanoma resection until data cut-off (September 25th, 2019) (range 38-64 months from initial surgery). Patient 3 had a local soft tissue recurrence at 26 months after initial melanoma resection, Pt. 5 experienced recurrence of an isolated lung metastasis at 40 months, which was resected followed by adjuvant therapy with nivolumab; at 53 months, several new lung metastases were detected. Pt. 1 developed multiple brain metastases at 40 months and underwent surgical resection of the dominant lesion. This was followed by multiple lines of therapy ultimately resulting in clinical stability at 64 months. Green line dabrafenib and trametinib (dab/tram) targeted therapy; NED, no evidence of disease. b. PBMCs from Pts. 11 and 12 were tested ex vivo by IFN-γ ELISpot against assay peptide (ASP) pools PA-PD in triplicate wells at each time point (error bars, standard error of the mean [s.e.m].; see IFN-γ ELISpot assay in Methods for statistical analysis). 2x105 PBMCs were plated with 5 μg/ml peptide and incubated overnight and normalized results are presented as previously as sfu/1 x 106 cells.

Figure 2.. Transcriptional profile of neoantigen-specific T cells over the course of vaccination.

a. Representative plots of ex vivo MHC class II tetramer staining of Pt. 4 (mut-ARHGAP29) CD4+ T cells at a series of time points pre- and following vaccination. Flow plots were pre-gated on CD4+ T cells. b. Kinetics of ex vivo tetramer-specific CD4+ T cell frequencies (mut-RUSC2, -ADAMT27, -ARHGAP29, and -ZNF281 tetramers for Pts. 1, 3, 4, and 5, respectively) following vaccination. c. Dose-response curves of CD137 (4-1BB) upregulation following neoantigen stimulation of selected TCR clonotypes from Patient 3, engineered into allogeneic T cells. These TCRs represented clonotypes that were observed in more than one cell (either as replicates at a single timepoint or at more than one timepoint) or as singletons (observed as a single replicate at a single timepoint). d. Clustering of tetramer-specific CD4+ T cells for Pts. 3, 4 and 5, depicted by cluster, patient and time point, respectively. Across these 3 patients, neoantigen-specific CD4+ T cells from 5 or 6 post-vaccination time points were collected, with a median of 310 total cells collected per patient (range 297-378). Ninety one and 73 non-tetramer-specific CD4+ T cells from Pts. 4 and 5, respectively, were collected prior to vaccination for comparison. e. Heatmap of top and bottom 20 differentially expressed genes by cluster. Selected marker genes are labeled. Normalized expression values are based on TPM counts. f. Feature plots of selected cluster marker genes. Clusters of interest are indicated by colored lines. g. Proportions of tetramer-positive CD4+ cells in each cluster by time. See Supplementary Dataset 5 for differentially expressed genes.

Figure 3.. TCR repertoire kinetics of neoantigen-specific T cells in relation to vaccination.

Paired TCRα and β clonotypes of mut-ADAMT27 CD4+ T cells, mut-ARHGAP29-specific CD4+ T cells and mut-ZNF281-specific CD4+ T cells for Pts. 3, 4 and 5, respectively, collected at different time points post-vaccination. Additional colors from left to right indicate the first appearance of new TCR clonotypes (defined as having a unique TRAV, TRAJ, TRBV, TRBJ, CDR3α and CDR3β amino acid sequence) at individual time points. Across 5 to 6 time points, 183, 89 and 107 distinct clonotypes were identified from 266, 329 and 210 single cells for each patient, respectively, none of which were shared with 133 clonotypes observed in non-tetramer-sorted cells prior to vaccination from Pts. 4 and 5. Pie charts indicate the proportions of TCR clonotypes originating at the individual time point and each previous time point. Numbers of T cells and TCR clonotypes per time point are shown for clonotypes appearing at more than one time point. Paired TCRα and β clonotypes from non-tetramer selected cells from Pts. 4 and 5 prior to vaccination are not shown. See Supplementary Dataset 7 for TCR clonotypes.

Figure 4.. Neoantigen-specific TCR dynamics following vaccination and PD-1 inhibition.

a. Plots of MHC class II tetramer staining of Pt. 2 and 6 CD4+ T cells after in vitro stimulation with mut-ADM2 and mut-MLL peptides, respectively, at week 16 following vaccination and after anti-PD-1 therapy with pembrolizumab (week 89 for Pt. 2, week 72 for Pt. 6). Flow plots were pre-gated on CD4+ T cells. b. Single-cell TCR sequencing of Pt. 2 and 6 tetramer-specific CD4+ T cells reveals enrichment of particular clonotypes. Beige bars indicate TCR clonotypes present at both week 16 and following pembrolizumab; gray bars indicate new TCR clonotypes after pembrolizumab (Pt. 2: 17 clonotypes; Pt. 6: 11 clonotypes). Each TCR clonotype is annotated for antigen reactivity following insertion of the engineered TCRs into primary human T cells. c. Pie charts depict proportions of individual tetramer-specific TCRs at week 16 and post-pembrolizumab. Colored clonotypes mark the top ten TCR clonotypes at week 16 (by frequency). The top 10 clonotype colors are arranged clockwise based on the clonotype abundance observed at week 16. In the post-pembrolizumab pie charts, the colors correspond to the same clonotypes shown in the week 16 pie charts. Beige color indicates non-dominant week 16 TCR clonotypes that were also present following pembrolizumab; gray color indicates new TCR clonotypes after pembrolizumab. d. Overlap of single cell observed TCR clonotypes identified in Patient 6 week 16 and post-pembrolizumab (week 68) peripheral blood samples and alpha and/or beta TCR sequences observed in bulk TCR sequence data from the recurrent tumor sample from Patient 6 at week 24. Only TCR sequences that were identical across TCRV, TCRJ and the CDR3 were considered a match. The subset of the identified clonotypes with confirmed neoantigen reactivity is indicated. See Supplementary Dataset 7 for TCR clonotypes.

Figure 5.. Vaccine-induced neoantigen specific T cells persist over several years.

a. Representative IFN-γ ELISpot responses of Pt. 3 neoantigen-specific CD4+ T cells specific for 4 ASP (15- to 16-mer) peptides and neoantigen-specific CD8+ T cells specific for 2 EPT (9-10-mer) peptides at 16 weeks and 47 months post-vaccination, respectively (cells were stimulated with peptides in vitro prior to ELISpots). b. Proportion of neoepitope peptides stimulating T cell reactivity that persisted to 2-4.5 years (filled blue and red) of the total responses detected at 16 weeks (blue outline), and after anti-PD-1 therapy (Pts. 2 and 6, red outline). Note that the x-axis scaling is different for CD4 (left) vs. CD8 (right) to visualize the distribution of persistent and non-persistent responses.. c. CDR3α/β chains of tetramer-specific TCRα/β that were detected in the bulk TCR sequencing of PBMCs collected between 100 and 212 weeks after vaccination. Timepoints of earlier detections by single-cell and bulk TCR sequencing and percentages of TCRα and TCRβ chains in the long-term bulk population are also shown. d. Percentages of Pt. 3 CD4+ T cells secreting cytokines in response to pools of ASP peptides that had generated ex vivo CD4+ responses as measured by intracellular cytokine staining after ex vivo stimulation with ASP pools at 16 weeks and at 47 months after vaccination. Negative controls (unstimulated CD4+ T cells) have been subtracted. Pie charts depict PD-1 expression, T cell phenotypes, and secretion of individual cytokines among the cytokine-producing CD4+ T cells. Markers were selected to evaluate cytokine secretion (IFN-γ, IL-2, and TNF-α), activation (PD-1), naïve (blue, CD27+/CD45RA+), effector (red [not visible], CD27−/CD45RA+), central memory (purple, CD27+/CD45RA−), and effector memory (orange, CD27−/CD45RA−) T cell phenotypes.

Figure 6.. Vaccine-induced T cell responses spread to non-vaccine neoantigen and TAA epitopes.

a. Somatic mutations and tumor-associated antigens were identified by WES of melanoma and germline DNA and their expression was confirmed by tumor RNA-seq. Non-vaccine neoantigen and tumor-associated antigen (TAA) epitope spreading peptides were selected on the basis of HLA binding predictions (Methods). PBMCs were pre-stimulated in vitro with epitope spreading peptides and reactivity was confirmed by IFN-γ ELISpot. b. Left: Pt. 3 IFN-γ secretion of CD4+ T cells stimulated with 3 non-vaccine neoantigen peptides measured by ELISpot in triplicates pre-vaccination and at week 16. Right: IFN-γ secretion of neoantigen-specific CD4+ T cells tested across a range of concentrations of mutated and wildtype peptides. c. Top left: Pt. 2 IFN-γ secretion of T cells stimulated with 2 non-vaccine neoantigen peptides (solid lines) and 2 TAA peptides (dashed lines) as measured by ELISpot in triplicates at week 16 and week 89 (after anti-PD-1 therapy). CR indicates complete response. Bottom left: Representative IFN-γ ELISpot response of mut-AGAP3c-specific CD4+ T cells. Right: IFN-γ secretion of neoantigen-specific CD4+ T cells tested across a range of concentrations of mutated and wildtype peptides; TAA-specific CD8+ T cells against TAA peptides (lower panels), respectively. d. IFN-γ ELISpot responses of CD4+ T cells specific for non-vaccine neoantigens persist up to 3 years post-vaccination in Pts. 2 and 3. ELISpots were performed in triplicate wells/condition (error bars, s.e.m).