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Supplemental Methods and Figure Legends

Supplemental Figure 1. Three melanoma cell lines (CHL1, Mel888, and WM793) were treated with DMSO (0), 10uM, or 50uM of dabrafenib (BRAFi) for 3 hours. Whole cell lysates were prepared, and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 2. Mel888 cells were treated with vehicle (DMSO) or 50 uM of BRAFi for 3 hours. Following treatment, cells were stained with fluorescently-labeled antibodies specific for MHC-I (HLA-A,B,C), MHC-II (HLA-DR), PD-L1, or melanoma-associated chondroitin sulfate proteoglycan (MCSP), and analyzed by flow cytometry. Data shown are representative of at least three replicate experiments with similar results. * indicates p < 0.05; ns, not significant. Supplemental Figure 3. HLA-A2-transduced Mel888 melanoma cell lines were treated with vehicle DMSO (0), 10uM, or 50uM of BRAFi for 3 hours. Whole cell lysates were prepared and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 4. HLA-A2-transduced Mel888 or WM793 cells were treated with DMSO or MEKi for 3 hours. Following treatment, cells were stained with a fluorescently-labeled HLA-A2-specific antibody and analyzed by flow cytometry. These experiments were repeated at least four times with similar results. *** indicates p < 0.005; ns, not significant. Supplemental Figure 5. T2 cells were pulsed with titrated amounts of MART-1(27-35) peptide, washed, and then used as stimulator cells for MART1-specific CD8+ TILs. Supernatants were collected after 8 hours of co-culture and analyzed by ELISA to measure interferon-gamma (IFNgamma) release. Supplemental Figure 6. Percentage of intracellular IFNgamma-positive TILs following 3 hours of co-culture with DMSO- or BRAFi-treated HLA-A*0201-transduced Mel888 cells or MART-1(27-35) peptide-pulsed WM793 cells, as measured by flow cytometry. All data are representative of experiments performed at least 3 times with similar results. *, p

S1. Ectopic expression of HLA allotypes in melanoma cell line Mel888. S2. SLC45A2 expression is restricted to melanoma tumor cells. S3. SLC45A2 gene expression in Cancer Cell Line Encyclopedia (CCLE) cell lines. S4. Quantitative comparison of SLC45A2 gene expression in various normal tissues and cancers. S5. Generation of HLA-A*0201-restricted SLC45A2 antigen-specific CTLs from multiple normal donor PBMC. S6. SLC45A2-specific CTLs demonstrate lytic activity against uveal and mucosal melanoma cell lines. S7. IFN-gamma release by MDA-specific CTLs in response to titrated HLA-A*0201 restricted peptides. S8. Primary melanocytes express HLA-A*0201 but are only killed by SLC45A2 T cells after peptide pulsing. S9. SLC45A2-specific CTLs from different donors preferentially kill melanoma tumor cells over primary melanocytes. Supplemental Table S1.Human MDA gene primer pair sequences used for RT-PCR. Supplemental Table S2. SLC45A2-derived peptides detected by mass spectrometric analysis of melanoma cell lines. Supplemental Table S3. Confirmation of natural SLC45A2 peptide processing and presentation by HLA transduction. Supplemental Table S4.Sequences and predicted binding affinities of HLA-A2-restricted MDA peptides.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAFV600E has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAFV600E also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway–driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting. Cancer Immunol Res; 3(6); 602–9. ©2015 AACR.

Cytotoxic T lymphocyte (CTL)–based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient–derived cell lines, we identified a number of shared HLA class I–bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402–restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618–29. ©2017 AACR.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types., Cytotoxic T lymphocyte (CTL)-based immunotherapies can induce tumor regressions by targeting HLA class I-bound tumor-associated peptides. Here, the authors identified a peptide derived from Vestigial-like 1 (VGLL1) as a shared, potentially therapeutic CTL target expressed by multiple cancer types.

BackgroundNeoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination.MethodsWe report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone.ResultsOut of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3–4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV.ConclusionsThese results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

Cytotoxic T lymphocyte (CTL)–based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient–derived cell lines, we identified a number of shared HLA class I–bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402–restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618–29. ©2017 AACR.

Several studies have demonstrated that oncogenic BRAF(V600E) promotes T-cell suppression in melanoma by upregulating the transcription of a multitude of immunomodulatory chemokine and cytokine genes. BRAF(V600E) has now been shown to act even more directly to evade cytotoxic T-cell recognition, by driving rapid internalization of human leukocyte antigen (HLA) class I from the tumor-cell surface and its intracellular sequestration.

High resolution cryo-electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3-D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member in the spirochetal family. High resolution cryo-ET reconstructions provided the detailed structures of the cell envelope, which is significantly different from that of gram-negative bacteria. The 4 nm lipid bilayer of both outer and cytoplasmic membranes resolved in 3-D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located, cone-shaped structure at both ends of bacterium. Furthermore, 3-D subvolume averages of the periplasmic flagellar motors and filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Together, our findings provide the most detailed structural understanding of the periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and escape host immune responses.

Background Oncogenic MAP kinase pathway activating mutations have been shown to drive immune suppression in melanoma and colorectal cancers. In this study, we explored whether oncogenic EGFR mutations play an analogous role in non-small cell lung cancer (NSCLC), and whether EGFRi can relieve tumor-associated immune suppression. Methods Lung cancer cell lines expressing wild-type or mutated EGFR were treated with EGFRi, followed by collection of RNA and cell supernatants. In parallel, tumor biopsies from NSCLC patients receiving a personalized peptide vaccine with or without concurrent EGFRi treatment were obtained. RNAseq-based transcriptome profiles of tumor cell lines and patient tumor biopsies were compared to assess common gene signatures driven by EGFRi. Gene expression changes were confirmed at the protein level using Western blot, flow cytometry, and cytokine/chemokine Luminex. The impact of EGFRi on T-cell migration and tumor cell recognition by antigen-specific CD8+ T cells was also assessed. Results In addition to downregulating genes associated with cell proliferation, apoptosis and survival, EGFRi increased the transcription of genes associated with TNFa and TRAIL signaling, and antigen presentation. HLA class I protein upregulation was confirmed and correlated with increased recognition of tumor cells by cytotoxic T cells. Several chemokines and cytokines were up- or down-regulated following EGFRi treatment, and Luminex analysis confirmed changes to 10 of them. Migration assays demonstrated that chemotaxis of T cells towards EGFR-mutant cell supernatants increased in an EGFRi dose-dependent manner. Transcriptome profiling of tumor biopsies revealed similar gene expression changes in on-EGFRi treatment tumor samples. Furthermore, increased tumor immune cell infiltration observed in EGFRi-treated patient tumors was consistent with the upregulation of EGFRi signature chemokines at the tumor site. Conclusions These results provide evidence that EGFRi has the capacity to facilitate modulation of the tumor microenvironment to favor immune cell infiltration and promote T-cell mediated antitumor immunity. Legal entity responsible for the study The University of Texas MD Anderson Cancer Center. Funding Tianjin HengJia Biotechnology Development Co., Ltd. Disclosure F. Li: Shareholder/Stockholder/Stock options: Tianjin HengJia Biotechnology Development Co., Ltd.. G. Lizee: Advisory/Consultancy: HengJia Neoantigen Biotechnology (Tianjin) Co., Ltd.. P. Hwu: Advisory/Consultancy: Dragonfly Therapeutics; Advisory/Consultancy: GlaxoSmithKline; Advisory/Consultancy, Shareholder/Stockholder/Stock options: Immatics; Advisory / Consultancy: Sanofi; Research grant/Funding (institution): Genentech. L. Deng: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Q. Zou: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Y. Wang: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. R. Ataullakhanov: Full/Part-time employment: Bostongene. Llc. Bagaev: Full/Part-time employment: Bostongene. Llc. N. Kotlov: Full/Part-time employment: Bostongene. Llc. V. Svekolkin: Full/Part-time employment: Bostongene. Llc. N. Miheecheva: Full/Part-time employment: Bostongene. Llc. F. Frenkel: Full/Part-time employment: Bostongene. Llc. All other authors have declared no conflicts of interest.