Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells

Abstract

T cell immunity directed against tumor-encoded amino acid substitutions occurs in some melanoma patients. This implicates missense mutations as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as targets of antitumor immunity is lacking. Moreover, it remains unknown whether vaccination can augment such responses. We found that a dendritic cell vaccine led to an increase in naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-β usage and clonal composition. Our results demonstrate that vaccination directed at tumor-encoded amino acid substitutions broadens the antigenic breadth and clonal diversity of antitumor immunity.

Trial registration: ClinicalTrials.gov NCT00683670.

Fig. 1. Vaccine candidate identification and immune monitoring

(A) Distribution of somatic (exomic and missense) mutations identified in patients MEL21 and MEL38 metachronous tumors (anatomical location and date of collection indicated) and patient MEL218 tumor are shown. HLA-A*02:01-binding candidate peptides were in silico identified among AAS and expression of gene encoding mutated protein determined from cDNA capture data (Table S1-S3). Venn diagrams show expression, among metachronous tumors, of mutated genes encoding vaccine neoantigens. The identities of the 3 immunogenic neoantigens identified in each patient are depicted in diagrams; color coding identifies naturally occurring (red) and vaccine-induced (blue) neoantigens. (B) Immune-monitoring of neoantigen-specific CD8+ T cell responses. Results are derived from PBMC isolated before DC vaccination (Pre-vaccine) and at peak (Post-Vaccine). PBMCs were cultured in vitro in the presence of peptide and IL-2 for 10 days followed by HLA-A*02:01/AAS-peptide dextramer assay. This immune monitoring strategy allows the reliable detection, as well as, the assessment of replicative potential of vaccine-induced T cell responses (Fig S4A). Color coding according to (A), numbers within dot plots represent percent neoantigen-specific T cells in lymph+/CD8+ gated cells.

Fig. 2. Antigenic determinants recognized by vaccine-induced T cells

(A) Neoantigen-specific T cells recognition of AAS (closed circles) and WT (open circles) peptides was determined in a standard 4h ⁵¹Cr-release assay using peptide titrations on T2 (HLA-A*02:01) cells. Percent specific lysis of triplicates (mean ± standard deviation) is shown for each peptide concentration; spontaneous lysis was <5%. Results are shown at 10:1 E: T ratios for all T cell lines except TMEM48 F169L and CDKN2A E153K T cells which are shown at 60:1 E:T ratio. A representative experiment of two independent evaluations is shown. (B) Neoantigen processing and presentation. Neoantigen-specific T cells were co-cultured with DM6 expressing AAS- (closed rectangles) or WT- (closed circles) TMC in a 4h ⁵¹Cr-release assay. Open triangles represent lysis obtained with parental DM6 cells. Percent specific lysis of triplicates (mean ± standard deviation) is shown for each E:T ratio; spontaneous lysis was <5%. A representative experiment of two independent evaluations is shown.

Fig. 3. Processing and presentation of tumor neoantigens

(A) RP-HPLC fractionation of HLA-A*02:01 peptides eluted from the AAS-TMC expressing melanoma cell line (black trace) and the synthetic peptide mixture containing MEL218 neoantigen candidates (red trace), with fraction 50 indicated. (B) Extracted ion chromatogram of the parent ion with the theoretical m/z of 480.8156 (+2) in HPLC fraction 50 from the HLA-A*02:01 eluted peptides (blue) overlaid with the EXOC8 Q656P synthetic peptide (pink). MS/MS fragmentation pattern of (C) the EXOC8 Q656P ion eluted from HLA-A*02:01 identified as IILVAVPHV, and (D) the corresponding synthetic peptide. (E) Same as in (A), with fraction 44 indicated. (F) Extracted ion chromatogram of the parent ion with the theoretical m/z 524.2808 (+2) in HPLC fraction 44 from the HLA-A*02:01 eluted peptides (blue) overlaid with the PABPC1 R520Q synthetic peptide (pink). MS/MS fragmentation pattern of (G) the PABPC1 R520Q ion eluted from HLA-A*02:01 identified as MLGEQLFPL and (H) the corresponding synthetic peptide.

Fig. 4. Vaccination promotes a diverse neoantigen-specific T cell repertoire

(A) Summary of TCRβ clonotypes identified, using neoantigen-specific TCRβ CDR3 reference libraries (see Tables S6-S10), in CD8+ T cell populations isolated from PBMC obtained before and after vaccination. Each symbol represents a unique TCRβ sequence and its frequency (%) in pre- and post-vaccine samples. Wilcoxon-signed rank test was performed and p values indicated in figure. (B) TCRβ CDR3 sequence of clonotypes (Tables S6-S10) identified in pre- (black bars) and post- (white bars) vaccine CD8+ T cell populations for neoantigens TKT R438W (pre=5, post=84 clonotypes); SEC24A P469L (pre=9, post=61) and EXOC8 Q656P (pre=2, post =12). Frequency of each unique clonotype is reported as percentage of total read counts.