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

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Science  15 May 2015:
Vol. 348, Issue 6236, pp. 803-808
DOI: 10.1126/science.aaa3828

Giving antitumor T cells a boost

Mutations allow tumors to divide, escape death, and resist treatment. But mutations can also cause tumors to express mutant proteins, which could potentially be exploited to drive antitumor T cell responses. Carreno et al. report the results of a small phase I trial seeking to do just this (see the Perspective by Delamarre et al.). They vaccinated three patients with advanced melanoma with personalized dendritic cell–based vaccines designed to activate T cells specific for mutations in the patients' cancer. T cells specific for mutant peptides did indeed expand. A next step will be to determine whether this promising strategy improves patient outcomes.

Science, this issue p. 803; see also p. 760

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.

Melanoma genomes harbor somatic mutations that are caused by exposure to mutagens such as ultraviolet light (1, 2). Tumor missense mutations, translated into amino acid substitutions (AASs), may provide a form of antigens that the immune system perceives as foreign, which in turn elicits tumor-specific T cell immunity (36). To examine the immunogenicity of tumor-encoded AASs, we obtained the consent of three patients with stage III resected cutaneous melanoma (MEL21, MEL38, and MEL218) for genomic analysis of their surgically excised tumors. The three patients were then enrolled in a phase 1 clinical trial with autologous, functionally mature, interleukin-12p70 (IL-12p70)–producing dendritic cell vaccine (fig. S1) (7). All three patients had received prior treatment with ipilimumab (see supplementary materials). Exome sequencing was performed to identify somatic mutations in tumor samples (Fig. 1A). Multiple metachronous tumors were analyzed from patients MEL21 and MEL38 (tables S1 and S2). Tumor missense mutations, translated as AAS-encoding nonamer peptides, were filtered through in silico analysis to assess HLA-A*02:01 peptide-binding affinity (8), and expression of genes encoding predicted HLA-A*02:01 peptide candidates was determined by analysis of cDNA capture data (Fig. 1A) (9). Peptide candidates for experimental validation were selected according to the strategy described in fig. S2; HLA-A*02:01 binding was evaluated using the T2 assay (fig. S3) (10) and confirmed with the fluorescence polarization–based competitive peptide-binding assay of Buchli et al. (11). Seven AAS peptide candidates per patient were selected from validated HLA-A*02:01 binders (fig. S2 and table S4) for incorporation into a personalized vaccine formulation, along with the melanoma gp100-derived peptides G209-2M and G280-9V as positive controls for vaccination (7). The expression pattern of mutated genes encoding vaccine candidates is shown in Venn diagrams in Fig. 1A.

Fig. 1 Vaccine candidate identification and immune monitoring.

(A) Distribution of somatic (exomic and missense) mutations identified in metachronous tumors of patients MEL21 and MEL38 (anatomical location and date of collection indicated) and tumor of patient MEL218. HLA-A*02:01-binding candidate peptides were identified in silico among AASs; expression of genes encoding mutated proteins was determined from cDNA capture data (tables S1 to S3). Venn diagrams show expression, among metachronous tumors, of mutated genes encoding vaccine neoantigens. The identities of the three immunogenic neoantigens identified in each patient are depicted; 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 PBMCs isolated before dendritic cell vaccination (prevaccine) and at peak (postvaccine). 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 is same as in (A); numbers within dot plots represent percent neoantigen-specific T cells in lymph+/CD8+ gated cells.

To examine the kinetics and magnitude of T cell immunity to AAS peptides upon vaccination, we collected peripheral blood mononuclear cells (PBMCs) prior to vaccination and weekly thereafter. The CD8+ T cell response to each peptide was analyzed using a HLA-A*02:01/AAS-peptide dextramer assay after a single round of in vitro stimulation (fig. S4A) (7). Immune monitoring demonstrated that in each patient, T cell immunity to one AAS peptide could be detected in prevaccine PBMC samples after in vitro stimulation (MEL21, TMEM48 F169L; MEL38, SEC24A P469L; MEL218, EXOC8 Q656P) (Fig. 1B), although not directly from the blood (fig. S4B). Preexisting immunity to these three neoantigens was confirmed in ex vivo expanded, prevaccine-purified CD8+ T cells by means of dextramer assay (fig. S4B) and interferon (IFN)–γ production (12) (fig. S4C).

Vaccination augmented the T cell response to these neoantigens, with observed frequencies of 23% TMEM48 F169L+ CD8+ T cells, 64% SEC24A P469L+ CD8+ T cells, and 89% EXOC8 Q656P+ CD8+ T cells detected upon culture at the peak of response (Fig. 1B). Immune monitoring also revealed vaccine-induced T cell immunity to two additional neoantigens per patient: TKT R438W and CDKN2A E153K (55% and 12%, respectively) in patient MEL21; AKAP13 Q285K and OR8B3 T190I (47% and 42%, respectively) in patient MEL38, and MRPS5 P59L and PABPC1 R520Q (58% and 84%, respectively) in patient MEL218 (Fig. 1B). Two of the three patients, MEL21 and MEL218, had preexisting immunity to G209-2M and G280-9V peptides, as determined by the presence of gp100-specific T cells in prevaccine PBMC samples and their ex vivo expansion upon antigen stimulation (fig. S5B). Upon vaccination, these T cell responses were enhanced in patients MEL21 and MEL218 and revealed in patient MEL38 (fig. S5). No T cell immunity to the remaining 12 AAS peptides was detected. Overall, robust neoantigen T cell immunity was detectable as early as week 2 and peaked at week 8 to 9 after the initial vaccine dose (fig. S4A). Neoantigen-specific CD8+ T cells were readily identified by dextramer assay directly in postvaccine PBMC samples (fig. S4B) and memory T cells were detected up to 4 months after the final vaccine dose.

Analysis of T cell reactivity among the three patients indicated no preferential skewing toward AAS at specific positions in the peptide sequence—that is, toward TCR contact residues or primary anchor residues (13). Rather, in each patient, T cell immunity appeared to focus on the three AAS candidates exhibiting the highest HLA-A*02:01 binding affinity, whereas the remaining medium- to high-affinity peptides were nonimmunogenic (table S4) (8, 11). Immunogenic AAS peptides (Fig. 1A) were not preferentially derived from genes with high allelic frequency or expression levels (tables S1 to S3).

To characterize the function of vaccine-induced neoantigen-specific T cells, we established short-term expanded CD8+ T cell lines and confirmed their antigen specificity by dextramer assay (fig. S4B) (7, 12). Neoantigen-specific T cells displayed significant levels of cytotoxic activity at AAS peptide concentrations of 1 to 10 nM, a finding that is consistent with high-avidity T cell recognition of antigen (Fig. 2A). OR8B3 T190I–specific T cells could not discriminate between AAS and wild-type peptide when presented on T2 cells, whereas all of the remaining T cell lines showed clear specificity for AAS peptide sequences (Fig. 2A). Next, we sought to characterize the cytokine production profile of these T cells, as a previous report suggests that IL-12p70–producing dendritic cells promote type 1 CD8+ T immunity, which in turn correlates with increased clinical benefit (7, 14). Upon antigen stimulation, most vaccine-induced neoantigen-specific T cells produced high amounts of IFN-γ relative to IL-4, IL-5, and IL-13—a pattern that is indicative of a type 1 phenotype (fig. S6). However, SEC24A P469L–specific T cells exhibited a type 2–skewed phenotype (high IL-4, IL-5, and IL-13 levels relative to IFN-γ), and TMEM48 F169L–specific T cells showed a mixed phenotype (high IL-13 levels, but not higher IL-4 or IL-5 levels, relative to IFN-γ) (fig. S6).

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

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

We next transfected DM6, a HLA-A*02:01+ melanoma cell line (15), with tandem minigene constructs (TMCs) to evaluate neoantigen processing and presentation. Each minigene encoded an AAS, or the corresponding wild-type amino acid, embedded in 19 to 21 amino acids derived from the normal gene product (fig. S7A and table S5). TMCs also encoded the West Nile virus (WNV) SVG9 (16) and melanoma G280 (17) antigenic determinants as controls (figs. S5 and S7B). Seven of the nine immunogenic neoantigens—TMEM48 F169L, TKT R438W, CDKN2A E153K, SEC24A P469L, AKAP13 Q285K, EXOC8 Q656P, and PABPC1 R520Q—were processed and presented, as evidenced by cytotoxic activity (Fig. 2B) and IFN-γ production (fig. S7C) by corresponding neoantigen-specific T cells upon coculture with DM6 cells expressing AAS-encoding TMCs (AAS-TMCs). In contrast, neither cytotoxic activity (Fig. 2B) nor IFN-γ production (fig. S7C) was observed upon coculture of OR8B3 T190I– and MRPS5 P59L–specific T cells with DM6 cells expressing AAS-TMCs; this finding suggests that these neoantigens are not processed and presented from endogenously expressed protein. None of the neoantigen-specific T cells recognized wild type–encoding TMCs (Fig. 2B and fig. S7C).

On the basis of these findings and the immune monitoring results (Fig. 1B), the nine neoantigens identified in this study fall into three distinct antigenic determinant categories (18, 19). TMEM48 F169L, SEC24A P469L, and EXOC8 Q656P represent dominant antigens, as T cell immunity was detected prior to vaccination (naturally occurring) (Fig. 1B) and these neoantigens were processed and presented from endogenously expressed protein (Fig. 2B). TKT R438W, CDKN2A E153K, AKAP13 Q285K, and PABPC1 R520Q are characterized as subdominant antigens, as T cell immunity required peptide vaccination (Fig. 1B) and these neoantigens were processed and presented from endogenously expressed protein (Fig. 2B). Finally, OR8B3 T190I and MRPS5 P59L constitute cryptic antigens, because peptide vaccination elicited T cell immunity even though these neoantigens were not processed from endogenously expressed protein.

To validate neoantigen processing and presentation, we performed proteomic analysis on peptides eluted from soluble HLA-A*02:01 molecules isolated from melanoma cells expressing a TMC encoding AAS candidates from a tumor of patient MEL218 (18, 19). Reverse-phase high-performance liquid chromatography (RP-HPLC) was used to reduce the complexity and determine the elution profile of the pool of soluble HLA-A*02:01–restricted peptides presented by melanoma cells, as well as the synthetic AAS peptide mixture (Fig. 3, A and E). The fractions corresponding to each synthetic peptide were subjected to nanoscale liquid chromatography–mass spectrometry (nano LC/MS). Extracted ion chromatograms revealed the presence of an eluted peptide with a retention time within 2 min of synthetic EXOC8 Q656P peptide in fraction 50 (Fig. 3B). MS/MS fragmentation pattern comparison of the eluted and synthetic peptides ensured EXOC8 Q656P sequence identity and confirmed HLA-A*02:01 presentation of this dominant neoantigen (Fig. 3, C and D). A similar analysis of fraction 44 demonstrated the HLA-A*02:01 presentation of subdominant neoantigen PABPC1 R520Q (Fig. 3, E to H). Together, these results show that two of the seven neoantigens included in patient MEL218 vaccine, along with antigen controls WNV SVG9 and G280 (fig. S8), are processed and presented in the context of HLA-A*02:01 molecules.

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 mass/charge ratio (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). (C and D) MS/MS fragmentation pattern of the EXOC8 Q656P ion eluted from HLA-A*02:01 identified as IILVAVPHV (C) and the corresponding synthetic peptide (D). (E) Same as in (A), with fraction 44 indicated. (F) Extracted ion chromatogram of the parent ion with the theoretical m/z of 524.2808 (+2) in HPLC fraction 44 from the HLA-A*02:01 eluted peptides (blue) overlaid with the PABPC1 R520Q synthetic peptide (pink). (G and H) MS/MS fragmentation pattern of the PABPC1 R520Q ion eluted from HLA-A*02:01 identified as MLGEQLFPL (G) and the corresponding synthetic peptide (H).

Little is known about the composition and diversity of neoantigen-specific T cells (20, 21) or about the effect vaccination may have on these repertoires. To address this question, we generated reference T cell receptor–β (TCR-β) complementarity-determining region 3 (CDR3) sequence libraries (fig. S9 and tables S6 to S10) from short-term expanded sorted neoantigen-specific T cells (97 to 99% dextramer-positive; fig. S10) and used them to characterize neoantigen TCR-β clonotypes in purified CD8+ T cells isolated from pre- and postvaccine PBMC samples (2224). In prevaccination CD8+ T cell populations, as few as 1 and as many as 10 unique TCR-β clonotypes per neoantigen were identified (Fig. 4A). Vaccination increased the frequency of most existing prevaccine TCR-β clonotypes and revealed previously undetected clonotypes for all six neoantigens (Fig. 4A). For both dominant and subdominant neoantigens, the TCR-β repertoire was increased significantly after vaccination (Fig. 4). For example, 84 clonotypes representing 19 TCR-β families were detected for TKT R438W, 61 clonotypes representing 12 TCR-β families were detected for SEC24A P469L, and 12 clonotypes representing 8 TCR-β families were detected for EXOC8 Q656P (Fig. 4B). Thus, peptide vaccination with functionally mature dendritic cells may promote the expansion of a highly diverse neoantigen TCR repertoire.

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

(A) Summary of TCR-β clonotypes identified, using neoantigen-specific TCR-β CDR3 reference libraries (see tables S6 to S10), in CD8+ T cell populations isolated from PBMCs obtained before and after vaccination. Each symbol represents a unique TCR-β sequence and its frequency (%) in pre- and postvaccine samples; P values are indicated (Wilcoxon signed-rank test). (B) TCR-β CDR3 sequence of clonotypes (tables S6 to S10) identified in prevaccine (black bars) and postvaccine (white bars) 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.

Our results indicate that vaccination with high-affinity, patient-specific, tumor-derived mutant peptides augments T cell immunity directed at naturally occurring (dominant) neoantigens and expands the breadth of the antitumor immune response by revealing subdominant neoantigens (25). Vaccination against tumor neoantigens appears safe, as all three patients are alive and well with no autoimmune adverse events. Inclusion of subdominant neoantigens into any therapeutic strategy would be expected to exert pressure to reduce the selection of antigen loss variants, especially in the setting of clonal tumor evolution (26), which occurs with targeted agents such as BRAF inhibition in melanoma (27, 28). The revelation of a highly diverse TCR-β repertoire specific for dominant and subdominant neoantigens was surprising and points to a potentially rich pool of naïve tumor-specific T cells that remain ignorant unless activated by vaccination. The effect of prior ipilimumab exposure on the prevaccine T cell repertoire in the three patients reported is unknown. However, recent data (29) indicate that administration of a CTLA-4 monoclonal antibody can influence TCR repertoire diversity in patients; this finding suggests a therapeutic strategy of testing checkpoint inhibitors, including ipilimumab, together with neoantigen vaccine formulations in order to further improve clinical outcomes. The paradigm described above could be applied to other malignancies presenting high mutational burdens, such as lung, bladder, and colorectal cancers (1). Other categories of genomic alterations such as deletions, insertions, and frameshift mutations may also generate potential neoantigens; mining these neoantigens may be particularly relevant in malignancies with low mutational burden, such as leukemias (30). Personalized immunotherapies targeting private somatic tumor alterations may become feasible in the near future.

Supplementary Materials

www.sciencemag.org/content/348/6236/803/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S10

Tables S1 to S10

References (3136)

References and Notes

  1. Acknowledgments: We thank T. Hansen, D. Link, and J. DiPersio for critical review; T. Ley, M. Walter, and J. Welch for advice and helpful discussions; R. Austin, R. Demeter, W. Eades, S. McGrath, W. Swaney, T. Vickery, J. Walker, and T. Wylie for technical assistance; L. Cornelius, R. Fields, R. Aft, J. Moley, A. Schaffer, and J. Steel for clinical collaboration; A. Alyasiry, S. Kalathiveetil, I. King, M. Schellhardt, and C. Rush for clinical research support; and B. Fritz from Adaptive Biotechnologies for technical guidance. Supported by Barnes-Jewish Hospital Foundation, Siteman Cancer Frontier Fund, Our Mark on Melanoma (MOM) Foundation, Come Out Swinging (COS) Foundation, Blackout Melanoma Foundation, and National Cancer Institute grants P30 CA91842 and R21 CA179695 (B. Carreno, PI). We acknowledge the Genome Institute at Washington University School of Medicine for the sequencing production pipeline that generated data, established by National Human Genome Research Institute grant 5U54HG00307 (R. Wilson, PI). The Siteman Cancer Center provided the use of the Flow Cytometry Core, Clinical Trials Core, Tissue Procurement Core, and Biologic Therapy Core. The data presented in this manuscript are tabulated in the main paper and the supplementary materials. The raw exome and transcriptome sequence data are available on the Sequence Read Archive database: Bioproject PRJNA278450. The raw proteomic data are available at MassIVE database ID MSV000079125. Washington University and the authors (B.M.C., V.M., E.R.M., and G.P.L.) have filed a patent application (US62/050,195) related to the use of tumor somatic mutations for cancer immunotherapy entitled “Cancer Immunotherapy, Methods, Compositions and Uses Thereof.” We thank the patients and families for their dedicated participation in this study.
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