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Purpose:Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown.Experimental Design:We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC.Results:We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers.Conclusions:HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

Supplementary Figures S1-S4, Tables S4, S6.

GISTIC all lesions

Characteristics of breast cancers ascertained from Penn Medicine hospitals

Purpose: Immunotherapy has the potential to improve the dismal prognosis in pancreatic ductal adenocarcinoma (PDA), but clinical trials, including those with single-agent PD-1 or PD-L1 inhibition, have been disappointing. Our aim was to examine the immune landscape of PDA as it relates to aspects of tumor biology, including neoepitope burden.Experimental Design: We used publicly available expression data from 134 primary resection PDA samples from The Cancer Genome Atlas to stratify patients according to a cytolytic T-cell activity expression index. We correlated cytolytic immune activity with mutational, structural, and neoepitope features of the tumor.Results: Human PDA displays a range of intratumoral cytolytic T-cell activity. PDA tumors with low cytolytic activity exhibited significantly increased copy number alterations, including recurrent amplifications of MYC and NOTCH2 and recurrent deletions and mutations of CDKN2A/B. In sharp contrast to other tumor types, high cytolytic activity in PDA did not correlate with increased mutational burden or neoepitope load (MHC class I and class II). Cytolytic-high tumors exhibited increased expression of multiple immune checkpoint genes compared to cytolytic-low tumors, except for PD-L1 expression, which was uniformly low.Conclusions: These data identify a subset of human PDA with high cytolytic T-cell activity. Rather than being linked to mutation burden or neoepitope load, immune activation indices in PDA were inversely linked to genomic alterations, suggesting that intrinsic oncogenic processes drive immune inactivity in human PDA. Furthermore, these data highlight the potential importance of immune checkpoints other than PD-L1/PD-1 as therapeutic targets in this lethal disease. Clin Cancer Res; 23(12); 3129–38. ©2016 AACR.

Immunotherapy in pancreatic ductal adenocarcinoma (PDA) remains a difficult clinical problem despite success in other disease types with immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell therapy. Mechanisms driving immunosuppression and poor T-cell infiltration in PDA are incompletely understood. Here, we use genetically engineered mouse models of PDA that recapitulate hallmarks of human disease to demonstrate that CD40 pathway activation is required for clinical response to radiotherapy and ICB with αCTLA-4 and αPD-1. The combination of an agonist αCD40 antibody, radiotherapy, and dual ICB eradicated irradiated and unirradiated (i.e., abscopal) tumors, generating long-term immunity. Response required T cells and also short-lived myeloid cells and was dependent on the long noncoding RNA myeloid regulator Morrbid. Using unbiased random forest machine learning, we built unique, contextual signatures for each therapeutic component, revealing that (i) radiotherapy triggers an early proinflammatory stimulus, ablating existing intratumoral T cells and upregulating MHC class I and CD86 on antigen-presenting cells, (ii) αCD40 causes a systemic and intratumoral reorganization of the myeloid compartment, and (iii) ICB increases intratumoral T-cell infiltration and improves the CD8 T-cell:regulatory T-cell ratio. Thus, αCD40 and radiotherapy nonredundantly augment antitumor immunity in PDA, which is otherwise refractory to ICB, providing a clear rationale for clinical evaluation.Significance: Radiotherapy and αCD40 disrupt key links between innate and adaptive immunity, ameliorating resistance to immune checkpoint blockade in pancreatic cancer via multiple cellular mechanisms. Cancer Res; 78(15); 4282–91. ©2018 AACR.

MuSIC-SMG calculations

Supplemental Methods: Mouse strains and flow cytometry antibodies Supplemental Figure 1: Individual tumor kinetics of the unirradiated tumor in the PDA model. Tumor kinetics (mean and individual) of the irradiated tumor and survival in the B16-F10 melanoma model. Supplemental Figure 2: Individual tumor kinetics of the unirradiated tumor in antibody depleted mice (Cd4, CD8 and Ly6c) and knockout mice (Morrbid, Batf3, IFN gamma, CD40.) Mean tumor kinetics of the unirradiated tumor and survival in uMT and B220 knockout mice. Supplemental Figure 3: Mean tumor kinetics of the unirradiated tumor and survival in antibody depleted mice (IFNAR) and knockout mice (MyD88, TLR4, IFNAR, Sting.) Supplemental Figure 4: Flow cytometry plots for the top RF variables for the effect of RT Supplemental Figure 5: As for Supplemental figure 4, but for CD40 Supplemental Figure 6: As for supplemental Figure 4, but for the peak response Supplemental Figure 7: Heat map of the RF variables that predict response in irradiated and unirradiated tumors at 24 and 72 hours. Supplemental Figure 8: Histological samples of CD8 T cell infiltrate and SMA expression. Supplemental Figure 9: Graphical representation

PAAD patient info

Supplementary Figure 1. Cumulative distribution of cytolytic index between PAAD and STAD cohorts; Supplementary Figure 2. Enrichment of selected immune related gene sets in cytolytichigh PDA tumors; Supplementary Figure 3. Enrichment of immune related gene sets in cytolytic-high PDA Tumors; Supplementary Figure 4. Cytolytic index associates strongly with expression of Immunome gene sets; Supplementary Figure 5. Expression of copy number alteration-associated and MYC target genes are statistically enriched in cytolytic-low PDA tumors; Supplementary Figure 6. Tumor cellularity/purity estimates do not correlate with mutational load; Supplementary Figure 7. Tumor purity estimates for PDA, stomach adenocarcinoma (STAD) and lung adenocarcinoma (LUAD); Figure S8. No difference in tumor cellularity or purity between cytolytic subtypes in TCGA PAAD cohort; Figure S9. Cytolytic subtypes associate with neoepitope load in stomach (STAD) and lung adenocarcinoma (LUAD); Figure S10. Neoepitope burden does not differ across PDA subtypes; Figure S11. Correlation plots of individual Immune suppression index versus cytolytic index.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1 , encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]–expressing CD8 + CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]–expressing CD8 + T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7 -dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)–driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3 + CD8 + and toward TCF1 + CD8 + to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Purpose: Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown. Experimental Design: We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC. Results: We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers. Conclusions: HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion,98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.

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.

Despite improved methods for MHC affinity prediction, the vast majority of computationally predicted tumor neoantigens are not immunogenic experimentally, indicating that high-quality neoantigens are beyond current algorithms to discern. To enrich for neoantigens with the greatest likelihood of immunogenicity, we developed an analytic method to parse neoantigen quality through rational biological criteria across five clinical datasets for 318 cancer patients. We explored four quality metrics, including analysis of dissimilarity to the non-mutated proteome that was predictive of peptide immunogenicity. In patient tumors, neoantigens with high dissimilarity were unique, enriched for hydrophobic sequences, and correlated with survival after PD-1 checkpoint therapy in patients with non-small cell lung cancer independent of predicted MHC affinity. We incorporated our neoantigen quality analysis methodology into an open-source tool, antigen.garnish, to predict immunogenic peptides from bulk computationally predicted neoantigens for which the immunogenic "hit rate" is currently low.

Fewer than 2% of computationally predicted neoantigens are immunogenic, a proven barrier to the efficacy of neoantigen vaccines. Currently, strong MHC affinity is dominant among neoantigen selection criteria for targeted therapies. To enrich for neoantigens with the greatest likelihood of immunogenicity, we developed an analytic method to parse neoantigen quality through rational biologic criteria and applied this method across five clinical datasets for 318 cancer patients. We assessed absolute MHC affinity, pathogen homology, and a novel analysis of dissimilarity to the self-proteome. We hypothesized that sequences with high dissimilarity to wild-type sequences would be more likely to perceived as “non-self” by immunosurveilling T cells. In a validation dataset, dissimilarity enriched for immunogenic peptides with an odds ratio of 34.9 (95% CI: 30.9 to 39.5, p < 0.001). Pathogen homology enriched 3.9-fold (95% CI: 3.6 to 4.3, p < 0.001), whereas selection on the basis of strong MHC-binding affinity alone enriches only 1.4-fold (95% CI: 1.2 to 1.5, p < 0.001). In patient tumors, these newly classified high-dissimilarity neoantigens were rare (1.18% of predicted MHC binders), did not overlap with the other metrics of neoantigen quality, and were enriched for hydrophobic sequences. While conventionally predicted neoantigens did not correlate with survival in two trials of PD-1 checkpoint therapy in patients with non-small cell lung cancer, high-dissimilarity neoantigens, even independent of MHC affinity, were predictive (HR - 3.29, 95% CI: 1.50 – 7.22, p = 0.014 and 3.40, 95% CI: 1.37 – 8.47, p = 0.021). Furthermore, we found that dissimilar peptides were enriched among known immunogenic neoantigens reported in multiple independent settings, including vaccine clinical trials (p = 0.001 – 0.023). This enrichment was not explained by a correlation with reported MHC binding affinities. Dissimilarity also enriched for immunogenicity in a dataset of human autoantigens, tumor-associated antigens, and cancer-testis antigens (odds ratio: 10.44, p < 2.2e-16). Dissimilarity to the self-proteome is therefore an orthogonal approach to MHC affinity for predicting CD8 T cell immunogenicity in both neoantigens and self-antigens. Overall, we show that neoantigen quality, including our novel analysis of dissimilarity to the self-proteome, identifies immunogenic peptides from conventionally predicted neoantigens for which the immunogenic “hit rate” is currently low and facilitates the more rapid development of neoantigen vaccines for cancer immunotherapy. Our pipeline, antigen.garnish, is open source and available on GitHub. This abstract is also being presented as Poster B30. Citation Format: Lee P. Richman, Andrew J. Rech, Robert H. Vonderheide. Emergence of quality metrics for neoantigens: Dissimilarity to the self-proteome as a novel determinant of immunogenicity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR14.

Immunotherapy in pancreatic ductal adenocarcinoma (PDA) remains a difficult clinical problem despite success in other disease types with immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell therapy. Mechanisms driving immunosuppression and poor T-cell infiltration in PDA are incompletely understood. Here, we use genetically engineered mouse models of PDA that recapitulate hallmarks of human disease to demonstrate that CD40 pathway activation is required for clinical response to radiotherapy and ICB with αCTLA-4 and αPD-1. The combination of an agonist αCD40 antibody, radiotherapy, and dual ICB eradicated irradiated and unirradiated (i.e., abscopal) tumors, generating long-term immunity. Response required T cells and also short-lived myeloid cells and was dependent on the long noncoding RNA myeloid regulator Morrbid. Using unbiased random forest machine learning, we built unique, contextual signatures for each therapeutic component, revealing that (i) radiotherapy triggers an early proinflammatory stimulus, ablating existing intratumoral T cells and upregulating MHC class I and CD86 on antigen-presenting cells, (ii) αCD40 causes a systemic and intratumoral reorganization of the myeloid compartment, and (iii) ICB increases intratumoral T-cell infiltration and improves the CD8 T-cell:regulatory T-cell ratio. Thus, αCD40 and radiotherapy nonredundantly augment antitumor immunity in PDA, which is otherwise refractory to ICB, providing a clear rationale for clinical evaluation. Significance: Radiotherapy and αCD40 disrupt key links between innate and adaptive immunity, ameliorating resistance to immune checkpoint blockade in pancreatic cancer via multiple cellular mechanisms. Cancer Res; 78(15); 4282–91. ©2018 AACR.

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 tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

The immune system exerts antitumor activity via T cell–dependent recognition of tumor-specific antigens. Although the number of tumor neopeptides—peptides derived from somatic mutations—often correlates with immune activity and survival, most classically defined high-affinity neopeptides (CDNs) are not immunogenic, and only rare CDNs have been linked to tumor rejection. Thus, the rules of tumor antigen recognition remain incompletely understood. Here, we analyzed neopeptides, immune activity, and clinical outcome from 6,324 patients across 27 tumor types. We characterized a class of “alternatively defined neopeptides” (ADNs), which are mutant peptides predicted to bind MHC (class I or II) with improved affinity relative to their nonmutated counterpart. ADNs are abundant and molecularly distinct from CDNs. The load of ADNs correlated with intratumoral T-cell responses and immune suppression, and ADNs were also strong predictors of patient survival across tumor types. These results expand the spectrum of mutation-derived tumor antigens with potential clinical relevance. Cancer Immunol Res; 6(3); 276–87. ©2018 AACR.

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.

In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through “immunoediting,” whereby tumors adapt to immune pressure and escape T cell–mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell–depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model — a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors.

Purpose: Immunotherapy has the potential to improve the dismal prognosis in pancreatic ductal adenocarcinoma (PDA), but clinical trials, including those with single-agent PD-1 or PD-L1 inhibition, have been disappointing. Our aim was to examine the immune landscape of PDA as it relates to aspects of tumor biology, including neoepitope burden. Experimental Design: We used publicly available expression data from 134 primary resection PDA samples from The Cancer Genome Atlas to stratify patients according to a cytolytic T-cell activity expression index. We correlated cytolytic immune activity with mutational, structural, and neoepitope features of the tumor. Results: Human PDA displays a range of intratumoral cytolytic T-cell activity. PDA tumors with low cytolytic activity exhibited significantly increased copy number alterations, including recurrent amplifications of MYC and NOTCH2 and recurrent deletions and mutations of CDKN2A/B. In sharp contrast to other tumor types, high cytolytic activity in PDA did not correlate with increased mutational burden or neoepitope load (MHC class I and class II). Cytolytic-high tumors exhibited increased expression of multiple immune checkpoint genes compared to cytolytic-low tumors, except for PD-L1 expression, which was uniformly low. Conclusions: These data identify a subset of human PDA with high cytolytic T-cell activity. Rather than being linked to mutation burden or neoepitope load, immune activation indices in PDA were inversely linked to genomic alterations, suggesting that intrinsic oncogenic processes drive immune inactivity in human PDA. Furthermore, these data highlight the potential importance of immune checkpoints other than PD-L1/PD-1 as therapeutic targets in this lethal disease. Clin Cancer Res; 23(12); 3129–38. ©2016 AACR.

Tumors with germline or somatic BRCA1 or BRCA2 alterations are sensitive to DNA-damaging agents but often develop therapeutic resistance. Breast cancers associated with BRCA1/2 alterations have a relatively high mutational load, defined as the number of nonsynonymous single nucleotide mutations, relative to BRCA1/2 wild-type tumors, leading to suggestions that patients with BRCA1/2 mutations are possible candidates for immune checkpoint blockade. However, immune infiltration can range widely in tumors with BRCA1/2 alterations, and molecular determinants that account for this variability remain unclear. Our goal was to delineate the molecular features associated with immunogenicity in breast cancers associated with germline or somatic BRCA1/2 alterations through genetic/genomic and histopathologic analyses in 115 tumors from the TCGA (n=89) and Penn (n=26). In the TCGA dataset, we found that the level of homologous recombination deficiency (HRD), as measured by the copy number-based HRD score (high vs. low, dichotomized by median), correlated positively with neoantigen load in BRCA1/2 tumors (p=0.02), but unexpectedly inversely with immunogenicity as measured by cytolytic index (p=0.04) (geometric mean of PRF1 and GZMA expression) and immune ESTIMATE (p=0.002), an RNA-seq-based measure of immune cell infiltration. In prior studies, we had found that a surprisingly high percentage of BRCA1 and BRCA2 germline mutation-associated breast cancers lacked allele-specific loss of heterozygosity (LOH), 10% and 46%, respectively. We thus evaluated whether allele-specific LOH of germline BRCA2 mutations and somatic BRCA1/2 mutation clonality were drivers of the negative association between HRD level and cytolytic index/immune ESTIMATE. We found that allele-specific LOH was associated with higher HRD score (p=0.015) but lower enrichment of the Type II IFN signaling immune metagene (p=0.01), lower cytolytic index (p=0.04), and lower immune ESTIMATE score (p=0.01); tumors with clonal somatic BRCA1/2 mutations had significantly higher HRD score (p=2.4E-07) and mutational burden (p=0.05) but lower cytolytic index (p=0.003) and immune ESTIMATE scores (p=5E-05) than tumors with subclonal somatic BRCA1/2 mutations. Stratifying by both hormone receptor expression and median HRD score, we found that triple-negative breast cancers with low HRD were the most immunogenic and hormone receptor-positive tumors with high HRD the least (p=0.001). We confirmed these TCGA findings by histopathology in tumors associated with germline mutations in BRCA1/2 from Penn. We found that CD45+ leukocyte (p=0.03) and CD8+ cytotoxic T-cell infiltration (p=0.03) as well as expression of the immune effector PRF1 (p=0.048) were significantly lower with high HRD. Further, tumors with allele-specific LOH of the germline BRCA1/2 mutation had significantly lower PRF1 staining (p=0.004), lower membrane levels of the immune checkpoint protein PDL1 (p=0.05), and lower CD8+ (p=0.001), FoxP3+ (p=0.03), and CD20+ (p=0.04) immune cells, suggesting an immunosuppressive role for allele-specific LOH leading to high HRD. These data are consistent with a prior pan-TCGA analysis demonstrating that increased tumor chromosome/arm level copy number alterations suppress immunogenicity. We have found, for the first time, that genomic instability in BRCA1/2 tumors is associated with lower antitumor immune activity, and that the immunogenicity of BRCA1/2 mutation-associated breast cancers is directly related to their HRD level (driven by allele-specific LOH and clonality) and receptor status, both clinically evaluable biomarkers. These findings have immediate implications for the stratification of patients with BRCA1/2 mutation-associated breast cancer for checkpoint blockade therapy. Citation Format: Adam A. Kraya, Kara N. Maxwell, Bradley Wubbenhorst, Brandon M. Wenz, John Pluta, Andrew J. Rech, Nicole Lunceford, Amanda Barrett, Nandita Mitra, Jennifer J.D. Morrissette, Anupma Nayak, Michael Feldman, Susan M. Domchek, Robert H. Vonderheide, Katherine L. Nathanson. Homologous recombination deficiency negatively predicts for immune infiltration and antitumor immune activity in breast tumors with BRCA1/2 alterations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A22.

Summary The biological and functional heterogeneity between tumors—both across and within cancer types—poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

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.

FoxP3( + ) CD4( + ) regulatory T cells (Tregs) are important mediators of peripheral immune tolerance, acting via multiple mechanisms to suppress cellular immunity including antitumor responses. Although therapeutic strategies have been proposed to deplete Tregs in patients with breast cancer and other malignancies, dynamic changes in the Treg compartment as a function of stage and treatment of breast cancer remain poorly understood. Here, we evaluated peripheral blood CD4(+) T cells and FoxP3(+) CD4(+) T cells from 45 patients with early or late stage breast cancer and compared percentages, absolute counts, and Treg function to those from healthy volunteers (HV) of comparable age. Patients having completed adjuvant chemotherapy and patients with metastatic cancer exhibited significantly lower absolute CD4 counts and significantly higher percentages of FoxP3(+) CD4(+) T cells. In contrast, the absolute counts of circulating FoxP3(+) CD4(+) T cells did not differ significantly among early stage patients, late stage patients, or HV. Functionally, FoxP3(+) CD4(+) T cells from all donor groups similarly expressed CTLA-4 and failed to secrete IFN-gamma in response to stimulation. Thus, although Tregs comprise an increased percentage of circulating CD4(+) T cells in patients with metastatic breast cancer and patients in remission after completing the adjuvant chemotherapy, the systemic Treg pool, as measured by absolute counts, appears relatively constant regardless of disease stage or treatment status. Total CD4(+) T cell counts are not constant, however, suggesting that homeostatic mechanisms, or susceptibility to cytotoxic or malignant insults, fundamentally differ for regulatory and non-regulatory CD4(+) T cells.

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition.

Ipilumimab, which is a monoclonal antibody to the inhibitory T-cell receptor CTLA-4, has been shown to improve survival in patients with metastatic melanoma; however, the response rate was only 10%, necessitating the need to develop ways to improve treatment. Anecdotal evidence suggests that stereotactic body radiation (SBRT) might cooperate with anti-CTLA4 to improve response rates both for irradiated and unirradiated tumors, a concept that is being tested in a phase I/II clinical trial as well as a pre-clinical mouse model. Our central hypothesis is that SBRT delivered to an index metastatic lesion will stimulate tumor antigen release and improve the anti-tumor immune response to antiCTLA4 in advanced melanoma. To pre-clinically model the combination of SBRT and anti-CTLA4, we are using a syngeneic transplantation model with B16-F10 melanoma, which is injected to the bilateral flanks. As expected, response of the primary tumor is generally dictated by whether it is irradiated or not. In the unirradiated secondary tumor, we have observed CD8+ T-cell dependent regression in the SBRT+anti-CTLA4 cohort resulting in improved progression-free survival (77%) compared to anti-CTLA4 alone (33%), SBRT alone (0%), or the control (0%). As not all mice respond to the combined therapy, we have also begun to interrogate potential mechanisms of resistance by trascriptomic profiling of resistant tumors. To test our hypothesis clinically, we have initiated a single-center, investigator-sponsored, phase I/II clinical trial of escalating doses of SBRT with ipilimumab in patients with metastatic melanoma. Patients undergo SBRT to a single metastatic deposit followed by standard-of-care ipilimumab. Two treatment strata are used: bone/lung vs liver/subcutaneous. To date, no dose-limiting toxicities have been observed in the first 12 patients enrolled on this trial. Major regressions of irradiated as well as non-irradiated lesions have been observed. Immune assessments to understand mechanism and resistance are underway. Thus, by understanding the genes and mechanisms associated with the SBRT +anti-CTLA4 response, our goal is to complement an on-going clinical trial with insight into potential biomarkers and therapeutic targets that may improve the selection and treatment of patients with metastatic melanoma.

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.

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. 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 and mPGES1 KOepi tumors. In the orthotopic mammary tumor model, 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 controls. Depletion of CD8+ cells alone, or together with CD4+ cells, significantly accelerated the growth of mPGES1 KD orthotopic tumors. 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 Vonderheide. mPGES1 deletion increases tumor susceptibility to immune suppression [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A094.

Regulatory T cells (T(regs)) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25(+) FoxP3(+) T(regs) in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration-approved CD25-blocking monoclonal antibody daclizumab with regard to human T(reg) survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25(high) CD45RA(neg) T(regs). Moreover, daclizumab-treated CD45RA(neg) T(regs) lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on T(regs) in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in T(regs) in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms T(regs) in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated T(regs) while avoiding autoimmunity.

CD4(+) regulatory T cells frustrate productive tumor immune surveillance and represent an obstacle for cancer immunotherapy. In mice, anti-CD25 antibody is an effective method of depleting CD25(+) FOXP3(+) T regulatory cells (Tregs) in vivo and enhancing cancer immunity. Here, we propose the use of the anti-CD25 monoclonal antibody daclizumab for the depletion of Tregs in cancer patients. In early results from an ongoing clinical trial, a single intravenous infusion of daclizumab in patients with metastatic breast cancer is associated with a marked and prolonged elimination of CD25(+) FOXP3(+) Tregs in peripheral blood. When a cancer antigen peptide vaccine is administered during the daclizumab-induced Treg nadir, effective generation of cytotoxic T lymphocytes has been observed, including those specific for neo-antigens, such as cytomegalovirus peptide used as an immunological control. If confirmed in additional patients, these observations suggest that daclizumab may be an effective and available therapeutic agent for Treg modulation in patients with cancer.

Purpose: Disturbed peripheral blood B-cell homeostasis complicates certain infections and autoimmune diseases, such as HIV and systemic lupus erythematosus, but has not been reported in cancer. This study aimed to investigate whether B-cell physiology was altered in the presence of melanoma and other cancers. Experimental Design: Flow cytometry was used to identify phenotypic differences in B cells from patients with melanoma and normal donors. In vitro stimulated B cells were assessed for responsiveness and also used as stimulators of allogeneic T cells in mixed lymphocyte reactions. Results: We show B-cell dysregulation in patients with advanced melanoma (n = 26) and other solid tumors (n = 13), marked by a relative and absolute loss of CD27+ (memory) B cells and associated with an aberrant systemic plasmacytosis. Functionally, B cells from patients with melanoma inefficiently up-regulated immunoregulatory molecules and weakly secreted cytokines in response to CD40 and toll-like receptor 9 agonists. Stimulated B cells from patients induced proliferation of alloreactive CD4+ T cells, but these T cells poorly secreted IFNγ and interleukin-2. These effects were recapitulated by using purified normal donor CD27neg B cells in these same assays, linking the predominance of CD27neg B cells in patients with the observed functional hyporesponsiveness. Indeed, B-cell dysfunction in patients strongly correlated with the extent of loss of CD27+ B cells in peripheral blood. Conclusions: Disturbed B-cell homeostasis is a previously unrecognized feature of patients with advanced melanoma and other cancers and may represent an unanticipated mechanism of immune incompetence in cancer.

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.

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 intrinsic and adaptive resistance through 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 (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of their T cell receptor (TCR) repertoire. Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion. 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 intrinsic and 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 present and targetable. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Joseph Benci, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Kathleen Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Stephen Hahn, E. 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 CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A056.

Background: Immune checkpoint inhibitors such as CTLA4 and PD-1 result in impressive clinical responses, but optimal results will require combination therapy. This raises fundamental questions about non-redundancy and mechanisms of resistance. Emerging data indicate that combining immune checkpoint inhibitors with radiation (RT) may hold promise. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. Methods: 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. Results: 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. Conclusions: 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, anti-CTLA4, and anti-PDL1 is a rational and non-redundant approach to improving resistance, response, and immunity to melanoma and other cancer types. Citation Format: Andrew J. Rech, Christina Twyman, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin D. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, George Xu, Michael Feldman, Tara C. Gangadhar, Stephen Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A52.

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

2508 Background: CD4+ regulatory T cells (Tregs) inhibit tumor immunity and represent an obstacle for cancer immunotherapy, but an effective strategy to deplete Tregs in pts has not been identified...