T-cell Cytolytic Activity Research Articles

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Ablation of RASA2 Improves T-cell Activity in Both Solid and Liquid Tumors

RASA2 ablation increases CAR T-cell cytolytic activity and improves persistent cancer cell killing.

Tumor Microenvironment Characterization in Breast Cancer and an Immune Cell Infiltration Score Development, Validation, and Application

The tumor microenvironment (TME) refers to the cellular environment in which tumors exist. An increasing number of reports have emphasized its role in tumor progression, prognosis, relapse, metastasis, and therapeutic response with breast cancer (BRCA). Few studies have revealed a systematic landscape of immune cell infiltration (ICI) in BRCA. In this study, we comprehensively analyzed the immune cells infiltrating TME in BRCA. Three ICI patterns were identified through an unsupervised clustering method and an ICI score was developed by a principal component analysis (PCA). A Kaplan-Meier survival with log-rank test revealed a significant overall survival (OS) difference of BRCA patients with these three ICI patterns. We also found that a high ICI score was characterized by an elevated tumor mutation burden (TMB), effector T-cell infiltration, INF-γ-related cytotoxicity, and cytolytic activity score. An independent cohort validated that this ICI score could be a prognostic indicator for BRCA. Two immunotherapeutic cohorts and two chemotherapeutic cohorts confirmed that patients with higher ICI scores showed significant chemotherapeutic and immunotherapeutic advantages. In summary, these results suggest that the ICI patterns could act as a prognostic indicator and that the ICI score could precisely predict the clinical outcome for BRCA patients.

Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition

ObjectivesEpigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate...

Potentiate Immune Activation and Function By Targeting Inhibitor of Apoptosis Proteins (IAPs) in Relapse/Refractory DLBCL

Background: The prognosis of relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) that had progressed after or are ineligible for high dose chemotherapy and autologous stem cell support (HDC-ASCS) is extremely poor (median overall survival of 6.3 months). Cellular based immunotherapy with Chimeric Antigen Receptor T-cell against CD19 (CART-19) has proven high effective in r/r DLBCL with an overall response rate of 52-83% and an overall survival rate of 40%. While r/r DLBCL benefits from CART-19 therapy, a significant number of progress after initial response stresses the need to improve the efficacy of this novel approach. A significant amount of work is being done in improving the manufacturing or activation of CART cells. However, there is few data on lymphoma associated factors that could influence response to CART-19 therapy. Previously we reported an imbalance and dysfunction of pro- and anti-apoptotic proteins, including Bak/Bax, Mcl-1/BCLxL/Survivin and upregulation of inhibitor of apoptosis (IAP) family proteins in rituximab+chemotherapy (R-chemo) resistant DLBCL. Targeting IAPs by small inhibitor LCL161 in lymphoma cells significantly overcame chemotherapy resistance in vitro and in vivo. LCL161 potentiated CART-19 cell cytotoxicity mediated in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma pre-clinical models. Here, we investigate the mechanism of action to enhance immune activation by targeting IAPs in r/r DLBCL. Methods: Primary T-cells were isolated and activated from peripheral blood apheresis samples from consenting adults (healthy human donors or patients with DLBCL) using Pan T-Cell Isolation Kit (MACS) and activated by T-cell Dynabeads human activator CD3/CD28 kit. Cells were expanded in RPMI media supplemented with IL2 for 2 weeks. At day 14, cells phenotype analysis was performed (CD3, CD4, CD8) by flow cytometry. T-cell number was calculated by trypan blue exclusion assay and proliferation was measured by presto blue assay after exposure to LCL161 or control for 48hrs. After exposure, CD4 and CD8 ratios were determined by cellular surface staining. T-cell function assay was determined by intracellular staining with perforin and granzyme B. Regulatory T-cells were determined as CD25+ and FOXP3+ staining cells. T-cell cytolytic activity was accessed by 51Cr release assay using 51Cr labeled rituximab-chemotherapy sensitive (Raji and RL cells) or resistant (Raji 4RH and RL 4RH) cell lines as target cells (effector/target ratio 10/1). To confirm the role of IAP proteins in the effects of LCL161 in T-cell activation, we conducted cytotoxicity assays using XIAP knock out lymphoma cell lines as target cells. Stable XIAP knock out cell lines were generated by CRISPR-Cas9 gene editing system. Lentiviral Cas9 nuclease particles were transduced into Raji and Raji 4RH cells followed by lentiviral XIAP sgRNA transduction. Lentiviral sgRNA non-targeting control was also used as negative control. Result: In vitro exposure of T-cells to LCL161 did not affect cell viability. On the other hand, it led to an increase in CD8+ T-cells and in the CD8:CD4 ratio. In addition, the percentage of regulatory T cells (CD25+ and FOXP3+) was reduced following LCL161 exposure. Using the T-cell function assay, we found that perforin and granzyme B inside T-cell was significantly increased post LCL161 exposure. The presence of LCL161 significantly improved the T-cell killing activity towards rituximab-chemotherapy sensitive and more importantly resistant cell lines. XIAP knock out in target cells resulted in abrogation of the enhance cytotoxicity exhibited by T-cells exposed to LCL161. Conclusion: Our data suggest that besides its previously documented direct anti-tumor activity, LCL161 has dual functions modulating immune cells function. LCL161 improve T-cell function by increasing the number of CD8+ T-cells and intracellular levels of granzyme B and perforin leading to an increase in T-cell cytotoxicity. In addition, LCL161 decreases the number of T-reg cells further enhancing immune activation. Taking together, targeting IAPs would be an attractive approach that potentiate current CART-19 immunotherapy in the clinical setting. Disclosures Hernandez-Ilizaliturri: Astra Zeneca: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Epyzome: Consultancy; Pharmacyclics: Consultancy; Amgen: Consultancy; Gilead: Consultancy.

Omics-wide quantitative B-cell infiltration analyses identify GPR18 for human cancer prognosis with superiority over CD20

Tumor-infiltrating B lymphocyte (TIL-B), and TIL-B-related biomarkers have clinical prognostic values for human cancers. CD20 (encoded by MS4A1) is a widely used TIL-B biomarker. Using TCGA-quantitative multiomics datasets, we first cross-compare prognostic powers of intratumoral CD20 protein, mRNA and TIL-B levels in pan-cancers. Here, we show that MS4A1 and TIL-B are consistently prognostic in 5 cancers (head and neck, lung, cervical, kidney and low-grade glioma), while unexpectedly, CD20 protein levels lack quantitative correlations with MS4A1/TIL-B levels and demonstrate limited prognosticity. Subsequent bioinformatics discovery for TIL-B prognostic gene identifies a single gene, GPR18 with stand-alone prognosticity across 9 cancers (superior over CD20), with further validations in multiple non-TCGA cohorts. GPR18's immune signature denotes major B-cell-T-cell interactions, with its intratumoral expression strongly tied to a “T-cell active”, likely cytolytic, status across human cancers, suggesting its functional link to cytolytic T-cell activity in cancer. GPR18 merits biological and clinical utility assessments over CD20.

Tumor fitness, immune exhaustion and clinical outcomes: impact of immune checkpoint inhibitors

Recently proposed tumor fitness measures, based on profiling neoepitopes for reactive viral epitope similarity, have been proposed to predict response to immune checkpoint inhibitors in melanoma and small-cell lung cancer. Here we applied these checkpoint based fitness measures to the matched checkpoint treatment naive Cancer Genome Atlas (TCGA) samples where cytolytic activity (CYT) imparts a known survival benefit. We observed no significant survival predictive power beyond that of overall patient tumor mutation burden, and furthermore, found no association between checkpoint based fitness and tumor T-cell infiltration, cytolytic activity, and abundance (tumor infiltrating lymphocyte, TIL, burden). In addition, we investigated the key assumption of viral epitope similarity driving immune response in the hepatitis B virally infected liver cancer TCGA cohort, and uncovered suggestive evidence that tumor neoepitopes actually dominate viral epitopes in putative immunogenicity and plausibly drive immune response and recruitment.

Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6−/− cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism

BackgroundTNF receptor family agonists and checkpoint blockade combination therapies lead to minimal tumor clearance of poorly immunogenic tumors. Therefore, a need to enhance the efficacy of this combination therapy arises. Antigen-presenting cells (APCs) present antigen to T cells and steer the immune response through chemokine and cytokine secretion. DRibbles (DR) are tumor-derived autophagosomes containing tumor antigens and innate inflammatory adjuvants.MethodsUsing preclinical murine lung and pancreatic cancer models, we assessed the triple combination therapy of GITR agonist and PD-1 blocking antibodies with peritumoral injections of DRibbles-pulsed-bone marrow cells (BMCs), which consisted mainly of APCs, or CD103+ cross-presenting dendritic cells (DCs). Immune responses were assessed by flow cytometry. FTY720 was used to prevent T-cell egress from lymph nodes to assess lymph node involvement, and MHC-mismatched-BMCs were used to assess the necessity of antigen presentation by the peritumorally-injected DR-APCs.ResultsTritherapy increased survival and cures in tumor-bearing mice compared to combined antibody therapy or peritumoral DR-BMCs alone. Peritumorally-injected BMCs remained within the tumor for at least 14 days and tritherapy efficacy was dependent on both CD4+ and CD8+ T cells. Although the overall percent of tumor-infiltrating T cells remained similar, tritherapy increased the ratio of effector CD4+ T cells-to-regulatory T cells, CD4+ T-cell cytokine production and proliferation, and CD8+ T-cell cytolytic activity in the tumor. Despite tritherapy-induced T-cell activation and cytolytic activity in lymph nodes, this T-cell activation was not required for tumor regression and enhanced survival. Replacement of DR-BMCs with DR-pulsed-DCs in the tritherapy led to similar antitumor effects, whereas replacement with DRibbles was less effective but delayed tumor growth. Interestingly, peritumoral administration of DR-pulsed MHC-mismatched-APCs in the tritherapy led to similar antitumor effects as MHC-matched-APCs, indicating that the observed enhanced antitumor effect was mediated independently of antigen presentation by the administered APCs.ConclusionsOverall, these results demonstrate that peritumoral DR-pulsed-BMC/DC administration synergizes with GITR agonist and PD-1 blockade to locally modulate and sustain tumor effector T-cell responses independently of T cell priming and perhaps through innate inflammatory modulations mediated by the DRibbles adjuvant. We offer a unique approach to modify the tumor microenvironment to benefit T-cell-targeted immunotherapies.

Flotetuzumab (FLZ), an Investigational CD123 x CD3 Bispecific Dart® Protein-Induced Clustering of CD3+ T Cells and CD123+ AML Cells in Bone Marrow Biopsies Is Associated with Response to Treatment in Primary Refractory AML Patients

Introduction The infiltration of immune cells into tumors has been associated with therapeutic effects in preclinical models and patients with cancer. In AML, we have previously reported that immune infiltrated TME is predictive of failure to cytotoxic chemotherapy, but associated with response to immunotherapy, specifically FLZ (Uy ASH 2018, Rutella ASH 2018). Furthermore, FLZ also affects immune infiltration in the TME (Rutella ASH 2018). NK cells play an important role in AML control (Ruggieri Science 2012). FLZ (MGD006/S80880) is a humanized DART® molecule that bridges CD123 on AML with CD3 on T cells and mediates anticancer activity via T-cell activation and cytolytic activity against the bound cancer cell. While this is well described in vitro, little evidence of this interaction is available in vivo. Methods Patients (pts) were treated on the recommended phase 2 dose (RP2D) of FLZ (multi-step lead-in dose followed by 500ng/kg/day, in 28-day cycles). We studied the bone marrow (BM) tissue samples for 6 primary refractory pts at baseline and after treatment. Response assessment was performed at day 25±3 days of each cycle. Serial BM samples were evaluated using 2 different staining panels (PD-L1, FoxP3, CD8, CD3, CD103 / CD123, CD3, CD57, CD16) on consecutive slides. Slides were stained using a Leica BondRx autostainer and fluorescence imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify CD3+ T cell clusters. Results Six pts with primary refractory AML were included in this report. Pts were heavily pretreated (median prior lines of therapy was 3, range 2-9), and had adverse cytogenetic risk (ELN 2017). Three pts had a complete remission (CR) after 1 cycle of therapy (CR, CRh, CRi), two went on the receive allogeneic stem cell transplant (HSCT). In baseline BM samples, CD3 and CD8 cell infiltrates were higher in CR vs non-responders (CD3+ 18.3% ±6.9 vs 9.3% ±1.8; CD8+ 9.4% ±3.5 vs 4.8% ±1.2; mean±SEM). Two of the three CR patients, who underwent HSCT, developed clusters (Figure 1) in their on-treatment biopsies with 65 and 22 clusters of an average of 34 and 17 T cells per cluster, respectively. All clusters in CR pts were found on or adjacent to CD123+ cells. The BM biopsy of the CR pt with no detected clusters had no unequivocal evidence of residual/recurrent leukemic blasts. This pt had their dose interrupted early due to non-treatment related AE (infectious complication) and did not receive a full cycle of treatment; the response was transient and the pt relapsed shortly thereafter. NK cells (CD57+CD16+) were increased in post treatment biopsies of CR vs non-responders (0.93 ±0.31 vs 0.27 ±0.13; mean±SEM) with the largest fold increase in CR (28 vs 9). Lastly, post treatment biopsy PD-L1 expression was higher in non-responders than CR (23% vs 16%) with non-responders exhibiting the largest fold change in total PD-L1+ cells (10.9 vs 2.2). Summary Consistent with its proposed mechanism of action, these data highlight for the first time, the dynamic induction of an increase in T-cell infiltration, and clustering around CD123 AML cells in the bone marrow microenvironment of two AML patients that responded to FLZ. In pts with resistance to FLZ (non-responders) PD-L1 induction was significantly higher indicating that in some pts treatment with sequential check point inhibitor could obviate this mechanism of resistance A trial combining FLZ with sequential administration of a PD-1 inhibitor (MGA012) is currently recruiting pts. Figure 1. Baseline and on-treatment IHC of BM biopsies of a FLZ-treated CR pt showing cluster formation following treatment. Disclosures Bifulco: Ventana: Other: advisory board; PrimeVax: Equity Ownership, Other: ScientificBoard; BMS: Other: Advisory Board; Providnece: Patents & Royalties: Imaging processing; Halio Dx: Other: advisory board. Wigginton:macrogenics: Employment, Equity Ownership; western oncolytics: Consultancy, Other: consultancy. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. Fox:Akoya: Research Funding; Bristol Myers Squibb: Research Funding; Definiens: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Research Funding; Ultivue: Membership on an entity's Board of Directors or advisory committees.

Cytolytic activity correlates with the mutational burden and deregulated expression of immune checkpoints in colorectal cancer

BackgroundMicrosatellite unstable colorectal cancers (MSI+ CRCs) expressing PD-L1, respond to anti-PD-1 or anti-PD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same. Our aim was to examine how the immune landscape relates to different aspects of the CRC’s biology, including neoepitope burden.MethodsWe used TCGA data to stratify patients based on a cytolytic T-cell activity expression index and correlated immune cytolytic activity (CYT) with mutational, structural, and neoepitope features of each tumor sample. The expression of several immune checkpoints was verified in an independent cohort of 72 CRC patients, relative to their MSI status, using immunohistochemistry and RT-qPCR.ResultsCRC exhibits a range of intertumoral cytolytic T-cell activity, with lower cytolytic levels in the tumor, compared to the normal tissue. We separated CRC patients into CYT-high and CYT-low subgroups. High cytolytic activity correlated with increased mutational load in colon tumors, the count of MHC-I/−II classically defined and alternatively defined neoepitopes, high microsatellite instability and deregulated expression of several inhibitory immune checkpoints (VISTA, TIGIT, PD-1, IDO1, CTLA-4, and PD-L1, among others). Many immune checkpoint molecules (IDO1, LAG3, TIGIT, VISTA, PD-1, PD-L1 and CTLA-4) expressed significantly higher in MSI+ CRCs compared to MSS tumors. The expression of Treg markers was also significantly higher in CYT-high tumors. Both individual and simultaneous high levels of CTLA-4 and PD-L1 had a positive effect on the patients’ overall survival. On the reverse, simultaneous low expression of both genes led to a significant shift towards negative effect. Assessed globally, CYT-low CRCs contained more recurrent somatic copy number alterations. PD-L1 protein was absent in most samples in the independent cohort and stained lowly in 33% of MSI CRCs. PD-L1+ CRCs stained moderately for CD8 and weakly for FOXP3. CYT-high colon tumors had higher TIL load, whereas CYT-high rectum tumors had higher TAN load compared to their CYT-low counterparts.ConclusionsOverall, we highlight the link between different genetic events and the immune microenvironment in CRC, taking into consideration the status of microsatellite instability. Our data provide further evidence that MSI+ and CYT-high tumors are better candidates for combinatorial checkpoint inhibition.

Abstract 3039: Targeting carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA)

Abstract Background: CEACAM6, a cell adhesion receptor of the Ig-like superfamily, interacts with other CEACAMs (1, 5, 7 & 8) and is over-expressed in human cancers including PDA. Over-expression of CEACAM6 causes resistance to anoikis - regulated apoptosis induced by inadequate or inappropriate cell-matrix interactions, promoting a TGFβ-mediated invasive phenotype. CEACAM6 as a predictive biomarker has not been fully investigated with regard to its potential as a candidate therapeutic target in PDA. We report a detailed analysis of CEACAM6 by expression profiling in several pancreatic tumor types, stromal cells, cell lines and mechanistic studies supporting CEACAM6 as a therapeutic target, when inhibited impacts several hallmarks key to PDA pathogenesis. Methods: RNAseq, expression arrays and clinical data was analyzed from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and GEO using R (v3.4.3). Expression profiles were analyzed across PDA subtypes, stroma types and overall survival. CRISPR/Cas9 knockout (KO) of CEACAM6 in HPAF-II cells was investigated by quantitative proteomics and genomics with corroborative signaling pathway analysis. A therapeutic monoclonal antibody scFv-Fc (IgG) targeting CEACAM6 was tested in an orthotopic mouse PDA model. Results: CEACAM6 is over-expressed in ~90% of primary and metastatic PDA samples, ~10-20 fold higher vs. normal cells. Basal and classical subtypes and activated stroma over-express CEACAM6, compared to low or normal stroma. TCGA dataset shows that PDA patients with high CEACAM6 expression have a poor prognosis vs. low expression. In addition, CEACAM6 over-expression is associated with mutant KRAS and immune suppression elaborated by low cytolytic T-cell activity. Correlation studies show that CEACAM6 is associated with genes involved in cell junctions and membrane integrity. HPAF-II cells with CEACAM6 KO showed changes to cell adhesion-extracellular matrix, transmembrane activity, increase in immune signaling and changes to enzymes of mitochondrial activity with significantly decreased mitochondrial membrane potential and ATP production. In addition, CEACAM6 KO cells have a significantly decreased growth rate with reduced Src, Akt and Fak signaling. The scFv-Fc has anti-PDA activity as a single agent in an orthotopic mouse model. Conclusions: CEACAM6 over-expression is a stroma-associated poor prognostic marker and a novel therapeutic target in PDA. The scFv-Fc targeting CEACAM6 shows promising anti-PDA activity and is being developed to enhance anoikis and restore host anti-tumor immunity. Citation Format: Ritu Pandey, Muhan Zhou, Shariful Islam, Baowei Chen, Paul Langlais, Anup Srivastava, Larry S. Cooke, Eric Weterings, Daruka Mahadevan. Targeting carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3039.

Effector T-cell cytolytic activity modules derived from CD3+ single cells from human primary triple-negative breast cancer (TNBC) in multiple solid tumors to predict response to immune checkpoint blockade therapy (ICB).

1073 Background: The prognostic and predictive value of tumor infiltrating lymphocytes for ICB has been recognized in a variety of tumor types, including TNBC. Nonetheless, our understanding of the mechanistic aspects of T cell activation remains incomplete. We hypothesize that a specific effector phenotype of T cell cytolytic activity (ECA) is a consistent feature of epithelial tumors, possibly varying by tumor types with a range of inflammatory features. Methods: We evaluated 6,311 purified CD3+ single cells from human primary TNBC and computed sample set enrichment scores of a set of previously published immune metagenes. Following unsupervised clustering of the enrichment scores of the entire single cell population, two subgroups of cells with highest and lowest average enrichment score of T cell cytolytic activity formed a basis for detecting functional gene expression modules. Spectral decomposition and Jackstraw analysis estimated eight modules with overlapping sets of genes. Each gene expression module was then used to train a Random Forest classifier of ECA phenotype. Results: We discovered that our module-derived classifiers were prognostic not only in TNBC samples obtained from both TCGA (N = 150) and METABRIC (N = 320) datasets but also in 14 other tumor types encompassing 6,000 samples. For example, patient samples from TCGA dataset predicted to be in group ECA ‘High’ have better progression-free survival (p-value: 0.0098l; HR: 0.30) and better overall survival (p-value: 0.0066; HR: 0.17). In both breast datasets, gene within the classifier are relatively under-expressed in ER+ tumors as opposed to HER2+ and TNBC (p-value < 2.2e-16). In a dataset of normal, pure DCIS and mixed DCIS (GSE26304;N = 114), the same genes were relatively under-expressed in DCIS samples relative to invasive tumors (p-value < 2.2e-16). Additionally, in a pre-therapy tumor dataset of fifty-one advanced melanoma patients treated with Nivolumab, who previously either progressed on ipilimumab or were ipilimumab-naïve, our module-derived classifier was able to classify responders and non-responders with 77% accuracy (p-value = 0.02) and was associated with progression-free survival (p-value = 0.03; HR: 0.28). Conclusions: Our study highlights one important application of single-cell genomics in our understanding of immune microenvironment and potentially identify new immunotherapy targets.

Immune Checkpoint Blockade for Advanced NSCLC: A New Landscape for Elderly Patients.

The therapeutic scenario for elderly patients with advanced NSCLC has been limited to radiotherapy and chemotherapy. Recently, a novel therapeutic approach based on targeting the immune-checkpoints has showed noteworthy results in advanced NSCLC. PD1/PD-L1 pathway is co-opted by tumor cells through the expression of PD-L1 on the tumor cell surface and on cells within the microenvironment, leading to suppression of anti-tumor cytolytic T-cell activity by the tumor. The success of immune-checkpoints inhibitors in clinical trials led to rapid approval by the FDA and EMA. Currently, data regarding efficacy and safety of ICIs in older subjects is limited by the poor number of elderly recruited in clinical trials. Careful assessment and management of comorbidities is essential to achieve better outcomes and limit the immune related adverse events in elderly NSCLC patients.

Epigenomic promoter alterations predict for benefit from immune checkpoint inhibition in metastatic gastric cancer

BackgroundUtilization of alternative transcription start sites through alterations in epigenetic promoter regions causes reduced expression of immunogenic N-terminal peptides, which may facilitate immune evasion in early gastric cancer. We hypothesized that tumors with high alternate promoter utilization would be resistant to immune checkpoint inhibition in metastatic gastric cancer.Patients and methodsTwo cohorts of patients with metastatic gastric cancer treated with immunotherapy were analyzed. The first cohort (N = 24) included patients treated with either nivolumab or pembrolizumab. Alternate promoter utilization was measured using the NanoString® (NanoString Technologies, Seattle, WA, USA) platform on archival tissue samples. The second cohort was a phase II clinical trial of patients uniformly treated with pembrolizumab (N = 37). Fresh tumor biopsies were obtained, and transcriptomic analysis was carried out on RNAseq data. Alternate promoter utilization was correlated to T-cell cytolytic activity, objective response rate and survival.ResultsIn the first cohort 8 of 24 (33%) tumors were identified to have high alternate promoter utilization (APhigh), and this was used to define the APhigh tertile of the second cohort (13 APhigh of 37). APhigh tumors exhibited decreased markers of T-cell cytolytic activity and lower response rates (8% versus 42%, P = 0.03). Median progression-free survival was lower in the APhigh group (55 versus 180 days, P = 0.0076). In multivariate analysis, alternative promoter utilization was an independent predictor of immunotherapy survival [hazard ratio 0.29, 95% confidence interval 0.099–0.85, P = 0.024). Analyzing tumoral evolution through paired pre-treatment and post-treatment biopsies, we observed consistent shifts in alternative promoter utilization rate associated with clinical response.ConclusionA substantial proportion of metastatic gastric cancers utilize alternate promoters as a mechanism of immune evasion, and these tumors may be resistant to anti-PD1 immune checkpoint inhibition. Alternate promoter utilization is thus a potential mechanism of resistance to immune checkpoint inhibition, and a novel predictive biomarker for immunotherapy.Trial RegistrationClinicalTrials.gov Identifier: NCT#02589496

RNA-seq for identification of therapeutically targetable determinants of immune activation in human glioblastoma.

We sought to determine which therapeutically targetable immune checkpoints, costimulatory signals, and other tumor microenvironment (TME) factors are independently associated with immune cytolytic activity (CYT), a gene expression signature of activated effector T cells, in human glioblastoma (GBM). GlioVis was accessed for RNA-seq data from The Cancer Genome Atlas (TCGA). For subjects with treatment-naïve, primary GBM, we quantified mRNA expression of 28 therapeutically targetable TME factors. CYT (geometric mean of GZMA and PRF1 expression) was calculated for each tumor. Multiple linear regression was performed to determine the relationship between the dependent variable (CYT) and mRNA expression of each of the 28 factors. Variables associated with CYT in multivariate analysis were subsequently evaluated for this association in an independent cohort of newly diagnosed GBMs from the Chinese Glioma Cooperative Group (CGCG). 109 TCGA tumors were analyzed. The final multiple linear regression model included the following variables, each positively associated with CYT except VEGF-A (negative association): CSF-1 (p = 0.003), CD137 (p = 0.042), VEGF-A (p < 0.001), CTLA4 (p = 0.028), CD40 (p = 0.023), GITR (p = 0.020), IL6 (p = 0.02), and OX40 (p < 0.001). In CGCG (n = 52), each of these variables remained significantly associated with CYT in univariate analysis except for VEGF-A. In multivariate analysis, only CTLA4 and CD40 remained statistically significant. Using multivariate modeling of RNA-seq gene expression data, we identified therapeutically targetable TME factors that are independently associated with intratumoral cytolytic T-cell activity in human GBM. As a myriad of systemic immunotherapies are now available for investigation, our results could inform rational combinations for evaluation in GBM.

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1–2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13–14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

Abstract 17: In situ immune expression signatures before and after ipilimumab therapy for relapsed acute myeloid leukemia after allogeneic stem cell transplantation

Abstract Immune evasion may facilitate hematologic relapse after allogeneic hematopoietic stem cell transplantation (allo-SCT). We recently demonstrated that CTLA4 blockade with ipilimumab reinvigorates a graft-versus-leukemia effect to induce complete remissions of acute myeloid leukemia (AML) relapsing after allo-SCT. Here, we hypothesized that immune subpopulation dynamics and activation states inferred from in situ gene expression changes within the leukemic microenvironment are informative of clinical outcome. We undertook an in situ transcriptomic characterization of paired, pre/post ipilimumab leukemic biopsies from five samples with relapsed, extramedullary AML. We performed a response-to-treatment expression analysis for each patient and found three immune patterns corresponding to three clinical response patterns following ipilimumab (durable response, relapse, and primary resistance). Durable responses were characterized by a diverse infiltration of activated cytotoxic T lymphocytes, B cells, and macrophages. In contrast, primary resistance was associated with both marked infiltration of CD8 T cells deficient in T-cell receptor signaling, activation, or cytolytic activity and absent infiltration of other immune effectors. Interestingly, the sample with transient response and eventual relapse demonstrated pre- and post-treatment immunologically “inflamed” states that were strongly downregulated during clinical relapse. In situ gene expression analyses of leukemic biopsies before and after ipilimumab therapy suggest that specific patterns of immune population dynamics and activation states underlie diverse clinical outcomes. Citation Format: Pavan Bachireddy, Matthew S. Davids, Michael S. Rooney, Michaela Bowden, Chensheng W. Zhou, Scott J. Rodig, Vincent Ho, Joseph Antin, John Koreth, Edwin Alyea, Robert J. Soiffer, Catherine J. Wu. In situ immune expression signatures before and after ipilimumab therapy for relapsed acute myeloid leukemia after allogeneic stem cell transplantation [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 17.

Immune Cytolytic Activity Stratifies Molecular Subsets of Human Pancreatic Cancer.

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.

Sipuleucel-T (sip-T) to induce cytolytic T lymphocyte (CTL) activity against target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

5046 Background: Sip-T is an FDA-approved autologous cellular immunotherapy for patients with asymptomatic or minimally symptomatic metastatic CRPC (mCRPC), manufactured from peripheral blood mononuclear cells (PBMCs) cultured with the immunogen PA2024 (prostatic acid phosphatase [PAP] conjugated to granulocyte macrophage colony-stimulating factor). Treatment with sip-T induces cellular and humoral immune responses to both PA2024 and PAP (Antonarakis, ASCO 2015; Petrylak, ECC 2015; Small Clin Cancer Res 2015). To further elucidate the mechanism of sip-T–induced immune responses, we evaluated proliferative and cytolytic characteristics of PA2024- and PAP-specific T cells. Methods: Antigen-specific proliferation of CD4 (T helper [TH]) and CD8 (CTL) T cells was quantified using flow cytometry. To assess CTL lytic activity, cell-surface CD107a expression, indicating lysosomal fusion with the cell membrane and expulsion of lytic enzymes, was measured after stimulation with PAP or PA2024. CTL lytic activity was assessed in 13 patients (from 3 sip-T trials [NCT01431391, NCT01981122, and NCT01487863] in hormone-sensitive PC and mCRPC patients) and 1 healthy donor (control). Results: In sip-T–treated patients, a significant increase in proliferation of both CTLs and TH cells was detected in response to PA2024 (p &lt; 0.05) and PAP (p &lt; 0.10) at week 6 compared with baseline. Increased PA2024-specific proliferation was maintained in TH cells through week 26 (p &lt; 0.05). CTL CD107a expression increased in sip-T patient samples stimulated with PAP and PA2024 but not in the healthy donor control. Preliminary analyses suggest that patients with longer overall survival (OS) may have greater CTL CD107a activity. Conclusions: Sip-T treatment was associated with well-maintained antigen-specific cellular responses, including robust CTL responses, demonstrating sip-T induces antigen-specific tumor lysis and providing a biologic basis for OS benefit in mCRPC patients. Ongoing work will examine whether patients with longer OS display greater CTL activity. Clinical trial information: NCT01431391; NCT01981122; NCT01487863.