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Neoantigen-based therapies hold the promise of being safe, personalized anti-cancer therapies for a broad range of cancers. A crucial step for the development of effective neoantigen therapies is the identification of putative tumor specific neoantigens typically achieved through in silico analyses. In silico-based approaches have the benefit of being fast, modular and reproducible. A wide range of tools are available for variant calling, determining peptide-HLA binding affinity, peptide foreignness, agritopicity and more, and researchers are continuously adding to the lot. However, there does not currently exist a common reference data set with which these different approaches can be compared, and the key parameters for effective neoantigen identification remain elusive. Here we introduce a consortium-based initiative, the Tumor nEoantigen SeLection Alliance (TESLA), to address these needs and describe straight-forward strategies through which the sensitivity and ranking ability of neoantigen prediction methods can be improved. TESLA participants were provided with whole exome and RNA sequencing from six tumors (3 melanoma and 3 NSCLC) and in turn submitted predictions for immunogenic peptides. Twenty-five groups from academia, pharma and biotech participated to this challenge. Peptides identification and ranking across the teams were assessed and showed little overlap. The limited overlap could not be explained simply by differences in variant calling. From these predictions, 608 peptides regrouping the teams' highly ranked epitopes plus epitopes frequently called and ranked across the teams, were assessed for T-cell recognition in patient-matched samples as well as for in-vitro MHC binding affinity. Analyses showed that peptides recognized by T-cells had significantly stronger MHC binding affinity and were derived from genes with significantly higher gene expression. Concomitantly, neoantigen pipelines which prioritized epitopes with strong binding affinity and/or which filtered out those originating from genes with low tumor variant allele fraction or low gene expression were associated with improved ability to identify and rank neoantigens. Direct interventions on participant predictions using these identified traits demonstrated substantial improvement in the performance of the neoantigen predictions. Pipeline analysis indicated that there are a range of approaches to neoantigen prediction and improving upon an existing neoantigen pipeline requires assessing that pipeline for a range of characteristics, including variant detection ability, peptide filtering ability, and peptide ranking ability, and improving those areas where the pipeline performance is sub-optimal. TESLA data will be continually available and serve as a living benchmark to improve neoantigen prediction. Citation Format: Daniel K. Wells, Kristen Dang, Vanessa M. Hubbard-Lucey, Kathleen C. Sheehan, Andrew Lamb, Jeffrey P. Ward, John Sidney, Ana B. Blazquez, Andrew J. Rech, Jesse Zaretsky, Begonya Comin-Anduix, Alphonsus H. Ng, William Chour, Thomas V. Yu, Hira Rizvi, Jia Chen, Patrice Manning, Taha Merghoub, Justin Guinney, Adam Kolom, Cheryl Selinsky, Antoni Ribas, Matthew D. Hellmann, Ton N. Schumacher, Nir Hacohen, Pia Kvistborg, Alessandro Sette, James R. Heath, Nina Bhardwaj, Fred Ramsdell, Robert D. Schreiber, Nadine A. Defranoux, TESLA Consortium. Strategies to improve the sensitivity and ranking ability of neoantigen prediction methods: Report on the results of the Tumor nEoantigen SeLection Alliance (TESLA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3210.

Pancreatic adenocarcinoma (PDA) is highly refractory to immunotherapy, a consequence of T-cell exclusion from the tumor microenvironment (TME). Based on a pathway analysis of human PDAs, we hypothesized that the receptor tyrosine kinase ephrin A2 (EPHA2) drives this immunosuppressive TME, as its expression negatively correlated with CD8A, CD3, PRF1, GZMB, and patient survival (EPHA2low/EPHA2high log rank hazard ratio 0.115, 95% CI of ratio 0.0315-0.416, TCGA dataset). Deletion of Epha2 in tumor cells increased T-cell influx, decreased the number of infiltrating myeloid suppressor cells, and sensitized tumors to therapy. Treatment of Epha2-deficient tumors with combination of chemo and immunotherapy resulted in suppressed tumor growth or tumor regression in up to 85% of cases. Examination of Epha2-dependent gene expression nominated Ptgs2 as a downstream mediator of T-cell exclusion. Like EPHA2, PTGS2 exhibited a negative correlation with intratumoral T cells, cytolytic activity, and patient survival (PTGS2low/PTGS2high log rank hazard ratio 0.152, 95% CI of ratio 0.054-0.430, TCGA dataset). KPCY mice (mutant KrasG12D (K), dominant negative p53R172H (P), Cre recombinase (C), YFP protein (Y)) deficient in pancreatic ductal cell Ptgs2 had significantly increased overall survival compared to Ptgs2 sufficient KPCY mice (Ptgs2def/Ptgs2suff log rank hazard ratio 0.5001, 95% CI of ratio 0.294-0.851). Ptgs2 deletion promoted T-cell influx in both autochthonous and implanted tumors. Inversely, overexpression of Ptgs2 decreased the number of tumor-infiltrating T cells, increased the proportion of suppressor myeloid cells, and conferred resistance to the combination therapy. Remarkably, pharmacologic inhibition of PTGS2 sensitized the tumors to immunotherapy, suppressing the growth of implanted tumors and increasing the survival of treated KPCY mice (median survival of untreated and treated mice 151 and 199 days, respectively; survival curve log rank p-value= 0.017). These studies suggest that a tumor cell-intrinsic EPHA2-PTGS2 signaling axis regulates the immune TME in PDA and suggests that a two-step approach targeting T-cell exclusion and exhaustion holds promise for this treatment-refractory disease. Citation Format: Nune Markosyan, Jinyang Li, Yu Sun, Lee Richman, Jeffrey Lin, Fangxue Yan, Liz Quinones, Yogev Sela, Taiji Yamazoe, Naomi Gordon, John Tobias, Katelyn Byrne, Andrew Rech, Garret FitzGerald, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2) in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B33.

Pathologic somatic gene mutations within the KRAS proto-oncogene occur in greater than 90% of pancreatic ductal adenocarcinomas (PDA). These mutations are often conserved involving the codon 12 position and most frequently result in G12D, G12V, or G12R transitions. Unfortunately, no mutant KRAS (mKRAS)-targeted therapies exist to date. We hypothesize conserved hot-spot KRAS mutations may serve as cancer neoantigens for targeted immune-based therapeutic approaches. Using a validated in silico epitope prediction pipeline, we identified mKRAS epitopes with predicted strong binding affinity to the highly prevalent MHC class I alleles HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. We then confirmed the processing, presentation, and binding of putative mKRAS epitopes using HPLC-tandem mass spectrometry and competitive peptide-binding assays. The immunogenicity of bona fide mKRAS epitopes was confirmed by the in vitro generation of mKRAS-specific CD8+ T cell responses using healthy donor blood samples that were restricted to HLA-A*03:01, -A*11:01, or -B*07:02 as determined by cytokine release assay and peptide/MHC multimer staining. Following T-cell receptor (TCR) sequencing, transgenic expression of mKRAS-specific TCRs on primary CD8+ T cells conferred cytotoxic potential against KRAS-mutated tumor cell lines expressing target HLA/mKRAS combinations. Importantly, mKRAS-specific CD8+ T cells did not cross-react with wild-type or alternatively mutated KRAS epitopes as measured by cytokine release or cytotoxicity assays. Based on these data, we initiated an adjuvant vaccine clinical study at the University of Pennsylvania Abramson Cancer Center targeting mKRAS in patients with resected PDA. The objective of this clinical study is to identify further mKRAS-specific TCR sequences for future adoptive T-cell therapy applications. To date, a total of 7 subjects have been enrolled, and 3 subjects have been vaccinated against targeted mKRAS short peptides utilizing a dendritic cell-based platform. Results from the first vaccinated subject have demonstrated the generation of a KRAS G12V-specific CD8+ T-cell response restricted to HLA-A*11:01 following vaccination with >10% mKRAS-specific T cells isolated following primary in vitro expansion of CD8+ T cells using antigen-pulsed dendritic cells as measured by peptide/MHC multimer staining. TCR sequencing has been completed and functional studies to validate antigen specificity and tumor cytotoxicity are in process. The results of these studies will serve to develop a widely applicable T-cell therapy for PDA patients, and will have implications for the large number of patients with other KRAS-mutated cancers. Citation Format: Adham S. Bear, Andrew J. Rech, Lee P. Richman, Mark H. O'Hara, Gerald P. Linette, Beatriz M. Carreno, Robert H. Vonderheide. Identification of T-cell receptors targeting mutant KRAS in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B04.

Despite the success of cancer immunotherapy in many disease types, pancreatic ductal adenocarcinoma (PDA) is notably unresponsive to immune checkpoint blockade (ICB) with αPD1 and/or αCTLA4. The mechanism of resistance is poorly understood, but tumor epitopes and the microenvironment, which is immunosuppressive and excludes T cells, are thought to be contributory. To improve response to ICB, we used subcutaneous and orthotopic murine models of PDA to investigate the effect of combination therapy with ICB (CTLA-4 and PD-1 antagonist antibodies), CD40 agonist antibody and radiation therapy (RT). Combination therapy with CD40 agonist antibody, ICB and RT resulted in decrease tumor burden, increase overall survival, and generation of long-term immunity. Response is dependent on T and short-lived myeloid cells, while it is independent of innate activation pathways. Together, these results suggest a dual role for both the innate and adaptive immune response in treating PDA. Citation Format: Hannah Dada, Andrew J. Rech, Christina Twyman-Saint Victor, Andy J. Minn, Robert H. Vonderheide. Radiotherapy and αCD40 non-redundantly augment immunity to checkpoint blockade in refractory pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2985.

Survival rates for pancreatic ductal adenocarcinoma (PDA) remain dismal. Immunotherapy has the potential to improve outcomes, but clinical trial results – particularly those employing single-agent CTLA4 or PD1/PD-L1 inhibition – have thus far been disappointing. We stratified human PDA based on a signature of cytolytic T-cell activity. Differences in cytolytic index correlated with defined PDA subtypes and distinct mutational events. Specifically, PDA tumors with low cytolytic activity exhibited increased genomic structural variation, including reproducible copy number alterations in MYC, FGFR1, NOTCH2, and CDKN2. In contrast to other tumors such as kidney cancer or melanoma, PDA tumors with a high cytolytic index lacked an increased mutation or neo-epitope load. Tumors with high cytolytic activity exhibited increased expression of several immune checkpoint genes, but PD-L1 expression was uniformly low. These data establish a link between genomic alterations and immune activation in PDA and suggest that immune checkpoints other than PD-L1/PD-1 may be attractive therapeutic targets in this lethal disease. Citation Format: David Balli, Andrew J. Rech, Ben Z. Stanger, Robert H. Vonderheide.{Authors}. Immune landscape of human pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA23.

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by adaptive resistance and prolonged interferon-gamma exposure, resulting in STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells, and macrophage depletion and/or PD-L1 blockade reverses T cell exhaustion. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of the T cell receptor (TCR) repertoire. RT with dual checkpoint blockade shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also evident. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Joseph Benci, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Steve Hahn, John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR05.

Optimal results with immune checkpoint inhibitors such as CTLA4 and PD-1 will likely require combination therapy. This raises important questions about tumor resistance and non-redundant mechanisms of action. Pre-clinical and clinical data indicate that radiation (RT) may augment responses to immune checkpoint inhibition. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. In a phase I clinical trial with 19 patients with multiple melanoma metastases, a single index lesion was irradiated with hypofractionated RT, delivered over two or three fractions, followed by four cycles of the anti-CTLA4 antibody ipilimumab. We reproduced this therapy in mice using the melanoma cell line B16-F10. For this, each flank of C57BL/6 mice was implanted with tumors to model multiple metastases. Mice received anti-CTLA4 (on days 5, 8, and 11), irradiation of one tumor using an image-guided micro-irradiator (20 Gy x 1 on day 8), or both treatments. Mechanistic studies were performed on material obtained from patients and mice at baseline and thereafter. Overall, treatment in the phase I study was well tolerated and toxicity was similar to that reported for anti-CTLA4. Major tumor regressions were observed in a subset of patients with metastatic melanoma treated with anti-CTLA4 + RT. In mice, although combined treatment enhanced the CD8 T cell to Treg ratios and improved responses in irradiated and unirradiated tumors, resistance was common. Genome-wide and unbiased analyses revealed that resistant tumors have increased PD-L1, interferon-stimulated genes, and exhausted T cells that depress the CD8/Treg ratio. Patients and mice with high PD-L1 tumors that were treated with RT + anti-CTLA4 poorly reinvigorated exhausted T cells, did not respond, and rapidly progressed. In mice, adding anti-PD-L1/PD-1 to RT + anti-CTLA4 reinvigorated exhausted T cells, leading to complete responses and immunity across multiple cancer types. The extent of T cell exhaustion/reinvigoration predicts response and can be assessed through peripheral blood. Resistance to RT + anti-CTLA4 results from depression in the CD8/Treg ratio due to elevated tumor PD-L1 and persistent T cell exhaustion. Both clinical and pre-clinical data suggest that the combination of RT with CTLA4 and PD-1 checkpoint blockade is a rational, non-redundant approach to overcoming tumor resistance and improving immunity in multiple cancer types. Citation Format: Andrew J. Rech, Christina Twyman-Saint Victor, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Joseph Benci, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin S. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi K. Amaravadi, Lynn M. Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael D. Feldman, Tara C. Gangadhar, Stephen M. Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual immune checkpoint blockade overcome tumor resistance and distinctly improve immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2015-2858

The pro-tumor contributions of prostaglandin (PG) E2 are established, as is the clinical efficacy of pharmacological inhibition of COX-2, the rate-limiting enzyme in PGE2 synthesis. In addition to the desired suppression of tumor PGE2, collateral loss of endothelial COX-2-derived PGI2 imposes a cardiovascular hazard that limits clinical use of COX-2 inhibitors. mPGES1, the terminal enzyme in the PGE2 synthesis pathway, is an alternative target to interrupt COX-2-driven events in tumors without elevating cardiovascular risk. We engineered mice transgenic for an activated HER2/neu oncogene to lack mPGES1 globally (mPGES1 KOgl) or only in mammary epithelial cells (mPGES1 KOepi). Abdominal mammary glands from wild type (WT), heterozygous (het), mPGES1 KOgl, and mPGES1 KOepi mice were harvested at 22 weeks of age, paraffin embedded, H&E stained and scanned for whole slide imaging (WSI). Tumor multiplicity was calculated based on the number of lesions on WSI. Adjacent sections were immunostained for Ki67, caspase 3, Factor VIII, CD3, and F4/80, to assess proliferation, apoptosis, angiogenesis, and tumor lymphocyte and macrophage infiltration. Tumor multiplicity was significantly higher in WT compared to either mPGES1 KOgl or mPGES1 KOepi mice. Concordantly, shRNA knock down (KD) of mPGES1 in mammary tumor cells dramatically suppressed their growth as orthotopic tumors in syngeneic immune competent WT hosts: 4 weeks after the injections, 6 out of 6 non-target shRNA control tumors grew successfully while 6 out of 10 mPGES1 KD cells failed to grow. In separate experiments, 14 of 14 non-target shRNA cells formed tumors after 2 weeks while 12 out of 18 mPGES1 KD tumors failed. By immunohistochemistry, no difference was observed in proliferation and apoptosis markers, as well as in the number of tumor infiltrating macrophages and lymphocytes, between WT, mPGES1 KOgl, and mPGES1 KOepi tumors. In contrast to our previous study in mice lacking epithelial COX-2 (COX-2 KOepi), vascularization was not different between mPEGS-1 KOepi and WT tumors suggesting divergence in the anti-tumorigenic mechanisms in mPEGS-1 KOepi and COX-2 KOepi tumors. By flow cytometry there was no difference in infiltrating macrophage and lymphocyte density or functional phenotypes, between WT, mPGES1 KOepi, and mPGES1 KD orthotopic tumors. Similarly, no difference was observed in the number of macrophages and myeloid derived suppressor cells in spleens from mPGES1 sufficient and mPGES1 KD orthotopic tumor bearing mice. In contrast, decreased co-inhibitory molecule PD-1 expressing CD4+ and CD8+ lymphocyte numbers were evident in draining lymph nodes in mice injected with mPGES1 KD cells compared to WT. This finding indicates that the absence of mPGES1-derived PGE2 renders tumor cells less efficient in creating and maintaining an immunosuppressive tumor microenvironment and that targeting mPGES1 may enhance anti-tumor immune mechanisms. Citation Format: Nune Markosyan, Andrew Rech, Robert H. Vonderheide, Garret A. FitzGerald, Emer M. Smyth. mPGES1 deletion and the mechanisms of tumor growth suppression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 469. doi:10.1158/1538-7445.AM2015-469