Cytotoxic T-cell Infiltration Research Articles

Abstract A22: Homologous recombination deficiency negatively predicts for immune infiltration and antitumor immune activity in breast tumors with BRCA1/2 alterations

Abstract 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.

EnLIGHTenment of tumor vessel normalization and immunotherapy in glioblastoma.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor. Despite aggressive standard care, GBM remains predominantly fatal; hence, new innovative therapies are required. Recent research published in the Journal of Pathology has identified the CGKRK peptide as a promising tool with which to specifically target the tumor vasculature from high-grade glioma. This tumor vessel-homing peptide was fused to the tumor necrosis factor superfamily member LIGHT/TNFSF14, and injected intravenously into murine orthotopic GBM models. After treatment, the tumor vasculature appeared to be less abnormal, with normalized features such as increased endothelial barrier integrity, pericyte contractility, and tumor perfusion. Moreover, CGKRK-LIGHT induced the appearance of high endothelial venules (HEVs), which are specialized structures that play a role in lymphocyte trafficking and have been shown to increase T-cell infiltration in solid tumors. Combining CGKRK-LIGHT with anti-angiogenic and immune checkpoint blockade treatments boosted HEV induction and cytotoxic T-cell infiltration, leading to a reduction in tumor burden. In this Commentary, I highlight the therapeutic opportunities provided by and the current limitations of LIGHT-vascular targeting peptide as a new approach to target GBM and enhance tumor vessel delivery and immunotherapy efficacy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 69: Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages

Abstract One of the most common treatment options for cancer is a chemotherapy. After chemotherapy, however, unwanted host effects provoke tumor recurrence and aggressiveness of cancer cells, which often arise as a consequence of disruption in the patient's immune system. Lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), which have capability of suppressing cancer progression, are depleted following chemotherapy. Conventional chemotherapeutic agents kill cancer cells, which boosts recruitment of myeloid cells. Tumor-associated macrophages (TAMs), emerging as abundant tumor-infiltrating myeloid cells in tumor microenvironment, play an important role in immunosuppression caused by conventional chemotherapy. Chemotherapy-induced dying cancer cells could hijack accumulated TAMs, provoking tumor recurrence. Therefore, reprogramming of TAMs to maximize the chemotherapeutic efficacy is considered a promising novel anticancer strategy. In this study, we investigated whether dying cancer cells after chemotherapy could reduce therapeutic efficacy by modulating tumor-infiltrating macrophage activity. In a 4T1 syngeneic mouse breast cancer model, there was a marked decrease in the proportion of M1-like TAMs expressing CD86 with tumor-suppressive activity and reduced tumor infiltration of CTLs following treatment with paclitaxel. Paclitaxel treatment also resulted in an enhancement of heme oxygenase-1 (HO-1) mRNA levels in tumor-infiltrating myeloid cells engulfing dying cancer cells. Consistent with the in vivo profile of TAMs, bone marrow-derived macrophages (BMDMs) phagocytosing dying breast cancer cells exhibited significant upregulation of HO-1 expression. HO-1 induction in BMDMs engulfing dying breast cancer cells facilitated M2 polarization. In contrast, abrogation of HO-1 activity sustained M1 activation of BMDMs co-cultured with dying breast cancer cells. Pharmacologic inhibition of HO-1 activity with zinc protoporphyin IX augmented paclitaxel efficacy by stimulating tumor infiltration of CTLs in a 4T1 breast cancer model. Furthermore, blockade of HO-1 in breast tumor-bearing mice induced increased M1-like TAMs expressing CD86 that plays an important role in stimulating antitumor immune response. Taken together, HO-1 induction by the chemotherapy-derived dying cancer cells in TAMs dampens antitumor effects of chemotherapy by manipulating innate and adaptive immunity. Our results demonstrate that targeting overexpressed HO-1 in the breast tumor microenvironment can control the immune system to potentiate the efficacy of chemotherapy. Citation Format: Seung Hyeon Kim, Xiancai Zhong, Shin-Young Gwak, Ishrat Aklima Muna, Sin-Aye Park, Young-Nam Cha, Young-Joon Surh. Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages [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 69.

Abstract 1023: Release of cancer cells from the prostate tumor microenvironment to promote neoantigen exposure and enhance anti-tumor immune response

Abstract Prostate tumors are widely considered to be immunologically silent and resistant to immunotherapy. PCa is widely considered to be an immune desert, with little infiltration of cytotoxic T lymphocytes (CTLs). However, anti-tumor immune responses have been seen in PCa in the right context. We believe we can boost immune response and T cell infiltration into PCa tumors by inhibiting the stromal derived factor 1-CXC chemokine receptor 4 (SDF1-CXCR4) signaling axis and releasing tumor cells from the immune-suppressive bone marrow niche. We have shown that CXCR4 antagonism causes PCa disseminated tumor cells (DTCs) to leave the bone marrow niche and enter the circulation as released circulating tumor cells (CTCs). Our hypothesis is that the release of PCa cells from the tumor microenvironment will enhance chemotherapy-induced cell death and the subsequent presentation of tumor-associated antigens, allowing increased anti-tumor response to immunotherapies. I generated a novel selection-free immunofluorescence-based protocol to confirm this in dissemination models of cancer in mice. After CXCR4 inhibition, I observed an approximate five-fold increase in the number of released CTCs in the blood of mice 24 hours after treatment. Very little research has been performed on the proliferation of CTCs or DTCs, particularly when released from the bone marrow niche. I have been able to mark proliferating cells in blood and bone marrow with EdU and quantify proliferating CTCs. We determined that while released cancer cells are proliferating more than DTCs, the ratio of released proliferating cells is not sufficient to cause a robust response to chemotherapy alone. This led to the hypothesis that released cells could provide an excellent substrate for the immune system. Evidence to support this idea is that we consistently observe the engulfment of CTCs by CD45-positive immune cells from the blood of mice, indicating that immune cells may respond to CTCs but not DTCs. Our approach is unique in that we are using CXCR4 inhibition to release cancer cells into the bloodstream to expose them to immune cells and initiate an immune response via activated CTL infiltration into PCa tumors. We believe this represents an innovative polyvalent autologous vaccine approach as it does not rely on a single antigen to stimulate CTLs, and also relies on each individual patient's cancer cells and immune cells. We have also generated some immunofluorescent protocols to determine the immune composition of circulating cells in relation to CTCs. While we believe that CTLs are responsible for most of the anti-tumor immune response, we are using this opportunity to discover novel immune cell types that may be present before and after therapy. This approach, when combined with classic cancer therapy and immunotherapy, may represent a novel way to boost response to cancer therapies in metastatic PCa. Citation Format: Kenneth C. Valkenburg, Kenneth J. Pienta. Release of cancer cells from the prostate tumor microenvironment to promote neoantigen exposure and enhance anti-tumor immune response [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 1023.

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [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 4966.

Abstract 4753: Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion

Abstract Tumors evade immune recognition and destruction by various established mechanisms, but less is known about how tumors short-circuit successful immune cell trafficking. To identify candidate proteins that inhibit immune trafficking, we developed a bioinformatics screen that utilizes an intratumoral cytotoxic T lymphocyte (CTL) gene signature that quantifies the relative abundance of tumor infiltrating CTLs. This gene signature was used as a stratification tool to examine differential gene expression between tumors of “low” versus “high” CTL status. We hypothesized that a subset of recurrently overexpressed genes in low-CTL tumors (i.e., immunologically “cold” tumors) may functionally act to limit lymphocyte infiltration into the tumor mass. A pan-TCGA analysis of 23 solid tumor types (n=8,875 tumors) revealed a number of recurrent genes highly significantly associated with low CTL infiltration. We found that 88.5% of tumor pairwise comparisons (among the 23 tumor types) showed significant overlap of top genes associated with low CTL status, consistent with the existence of conserved, tumor-agnostic mechanisms of immune evasion. From these analyses, the secreted TGFβ superfamily member, BMP7, emerged as a top-ranking candidate regulator of immune trafficking. To investigate the role of BMP7 in CTL tumor trafficking, an immunogenic variant of the 4T1 mouse breast cancer cell line, 4T1-S, was modified using CRISPR-Cas9 to inducibly express BMP7 from the ROSA26 locus. 4T1-S BMP7-inducible cells were orthotopically transplanted into Balb/C female mice and tumors were allowed to form with or without BMP7 induction. Mice were sacrificed at 2, 3, and 4 wks post-tumor inoculation, and tumors, tumor-draining LNs (TDLNs), and spleens were harvested. At both 2 wks and 3 wks, a BMP7-dependent reduction in tumor CTLs was observed by anti-CD8a IF microscopy (p=0.002 and p<0.001, respectively). Flow cytometric analysis confirmed the significant reduction in tumor-infiltrating CD8+ CTLs in the BMP7-induced group at 3 wks (p=0.02). BMP7 expression did not alter tumor trafficking of natural killer (NK) cells. Flow analysis also showed CTL reduction in TDLNs at 2 wks (p=0.02), and NKs were reduced in the TDLNs at 3 wks (p=0.04). Spleen sizes were also significantly smaller at all time points, indicating a systemic immunomodulatory function of tumor-derived BMP7 (p=0.002, p<0.001, and p=0.002 at 2, 3, and 4 wks, respectively). This study provides a framework for discovering yet unappreciated mechanisms by which tumors evade the immune system, and presents first evidence that BMP7 expression by breast tumors can function to limit CTL trafficking. Citation Format: Eric D. Routh, Julia Chifman, Ashok Pullikuth, Lance D. Miller. Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion [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 4753.

Abstract 4726A: The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model

Abstract Background: Hypoxia, a common feature in solid tumors such as glioblastoma (GB), is associated with resistance to chemo- and radio-therapies and poor patient outcomes. In addition, hypoxia promotes the immune escape of tumors. Therefore, reversing tumor hypoxia to create an immunopermissive microenvironment can improve antitumor response, and combined with immunotherapy approaches such as checkpoint inhibitors (CPI), may increase therapeutic efficacy. OMX is an oxygen carrier well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and efficiently accumulates in orthotopic rodent GB and spontaneous canine brain tumors. Consequently, OMX significantly reduces hypoxia and improves the efficacy of radiotherapy and CPI. Methods: We used in vivo bioluminescence imaging of tumor, immunohistochemistry, flow cytometry, and cytokine multiplex assays to evaluate OMX's ability to immunosensitize the GL261 brain tumor microenvironment and promote tumor cures. Results: A single dose of OMX in brain tumor-bearing mice reduces tumor hypoxia, enhances the recruitment and infiltration of tumor-specific CX3CR1+ CD8 T cells into the tumor (using the EphA2 as a GL261-specific tumor antigen), decreases Tregs and increases activation and proliferation of cytotoxic T lymphocytes (CTLs). Specifically, OMX increases the Teff/Treg ratio by ~3-fold, indicating a switch from an immunosuppressive to an immunopermissive microenvironment. Similarly, when combined with anti-PD-1, OMX decreases Tregs, increases CTL infiltration, proliferation and cytotoxic activity, and modulates IFNg and IFNg-inducible cytokines that polarize T cells towards a Th1 phenotype. Treatment with OMX alone resulted in a 55% tumor cure rate, comparable to anti-PD-1 treatment. Furthermore, in late-stage tumor-bearing mice, we observed a 40% tumor cure rate for the combination of OMX with anti-PD-1, while anti-PD-1 alone resulted only in 5% tumor cures. In symptomatic mice with very high tumor burden, in which the combination of anti-PD-1 with anti-CTLA4 does not provide tumor cures, the addition of OMX resulted in a 20% tumor cure rate. Following rechallenge with GL261 tumor cells injected on the other side of the brain, all mice treated with OMX alone or in combination with CPI survived, indicating the presence of long-term immunologic memory against glioma cells. The survival benefit observed with OMX could be predicted with an identified circulating chemokine biomarker signature (post-hoc test). Conclusion: By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anticancer immune responses in GB patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. Citation Format: Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, Carol Liang, Jonathan Winger, Stephen P. Cary, Nicholas Butowski, Ana Krtolica. The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model [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 4726A.

Anti-CD73 and anti-OX40 immunotherapy coupled with a novel biocompatible enzyme prodrug system for the treatment of recurrent, metastatic ovarian cancer

Approximately 75% of ovarian cancer is diagnosed once metastasis to the peritoneal cavity has occurred. A large proportion of patients eventually develop platinum-resistive tumors, which are considered terminal. In order to provide an alternative a novel fusion protein, mCTH-ANXA5, has been developed for the treatment of recurrent, metastatic ovarian cancer. The fusion protein combines annexin V (ANXA5), an ovarian tumor and tumor vasculature targeting protein, with mutated cystathionine gamma-lyase (mCTH), an enzyme that converts selenomethionine (SeMet) into toxic methylselenol, which generates reactive oxygen species and eventual tumor cell death. In order to further enhance the therapeutic efficacy, anti-CD73 and anti-OX40 immunostimulants were combined with mCTH-ANXA5, resulting in an increase of survival by 100% from 12 to 24 days post-therapy and decrease tumor burden in mice with orthotopic metastatic ovarian cancer. Further evaluation of the combination therapy revealed a strong antibody-mediated immune response, and an increased infiltration of cytotoxic T-cells along with a decrease in tumor promoting immune cells. This study demonstrates the efficacy of a synergistic, multi-drug system by attacking the tumor as well as enlisting the body's own defense system to treat the patient.

Abstract A108: DNA damage repair targeting upregulates PD-L1 level and potentiates the effect of PD-L1 blockade in small cell lung cancer

Abstract Background: Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, are urgently needed. SCLC has a relatively immunosuppressed phenotype with relatively low levels of infiltrating T-cells and reduced antigen presentation. Only a minority of SCLC patients responds to programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors as monotherapy. Therefore, there is a critical need to develop strategies to enhance the efficacy of immunotherapy in SCLC. We previously discovered that DNA damage repair (DDR) protein, checkpoint kinase 1 (CHK1), is overexpressed in SCLC; CHK1 inhibitor, LY2606368, has activity in preclinical models of SCLC, a finding that supported the phase 2 clinical trial of LY2606368 for SCLC patients (NCT02735980). We hypothesize that targeting CHK1 by inducing DNA damage can enhance immunogenicity, antitumor immunity, and response to immune checkpoint targeting. Methods: SCLC cell lines and immune competent murine models were treated with single agent LY2606368, anti-PD-L1, or combination. End point analyses were done by Western blot, real-time RT-PCR, multicolor flow cytometry, and reverse phase protein array (RPPA). Result: SCLC tumors with higher DDR mutation burden have a higher expression of PD-L1 (p<0.0001). LY2606368 treatment enhanced the protein (but not mRNA) and surface expression of PD-L1 in SCLC cell lines and tumor models. We further observed increased PD-L1 glycosylation post-LY2606368 treatment. LY2606368+/- anti-PD-L1 combination activates mTOR and inactivates GSK3β pathway, thus providing mechanistic insight into CHK1-targeting mediated PD-L1 glycosylation and stabilization. We treated tumor-bearing immune-competent B6129F1 mice with either IgG (control), LY2606368 (10mg/kg, 2 out of 7 days), anti-PD-L1 (300ug, 1 out of 7 days) or combination (n=10/group). At Day 21, anti-PD-L1 did not cause tumor regression and LY2606368 treatment delayed tumor growth [T/C=0.31(p<0.001)]. However, most notably, 60% mice treated with anti-PD-L1+LY2606368 had a complete tumor regression within 15 days of treatment [T/C=0.05(p<0.001)]. Cotargeting CHK1+PD-L1 significantly increases the level of CD8+ cytotoxic-T cell tumor infiltration, decreases exhausted T cell population, and enhances the expression of IFN gamma in SCLC models. CD8 depletion was able to reverse the antitumor effect of the combination, demonstrating the role of cytotoxic T-cell infiltration in CHK1+PD-L1 response. Significance: Our study demonstrates, for the first time, the potential for combining the two promising modalities, immune checkpoint targeting and CHK1 inhibition, in SCLC. This study further demonstrates the novel mechanism of DDR-mediated PD-L1 regulation in SCLC. The immune profiling and correlative biomarker data from our study further provide valuable mechanistic insight and indicate the subset of the patient population who are most likely to respond to these treatments. This study suggests intriguing possibilities for therapeutic synergy and scientific rationale for the evaluation of PD-L1/PD-1 blockade with CHK1i in clinical trials for SCLC, a disease with dismal 5-year survival rate of <5%, which has remained unchanged for >30 years. Citation Format: Triparna Sen, Limo Chen, B. Leticia Rodriguez, Carl M. Gay, Lerong Li, Yanli Li, Youhong Fan, Bonnie Glisson, Jing Wang, Helen Piwnica-Worms, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. DNA damage repair targeting upregulates PD-L1 level and potentiates the effect of PD-L1 blockade in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A108.

BRAF peptide vaccine facilitates therapy of murine BRAF-mutant melanoma.

Approximately, 50% of human melanomas are driven by BRAF mutations, which produce tumors that are highly immunosuppressive and often resistant to vaccine therapy. We introduced lipid-coated calcium phosphate nanoparticles (LCP NPs) as a carrier to efficiently deliver a tumor-specific antigen, the BRAFV600E peptide, to drive dendritic cell (DC) maturation and antigen presentation in C57BL6 mice. The BRAF peptide vaccine elicited a robust, antigen-specific cytotoxic T cell response and potent tumor growth inhibition in a murine BRAF-mutant melanoma model. Advanced BRAF-specific immune response was illustrated by IFN-γ production assay and cytotoxic T lymphocyte (CTL) assay. Remodeling of immunosuppressive modules within the tumor microenvironment further facilitated CTL infiltration. Thus, using LCP NPs to deliver the BRAF peptide vaccine is a promising strategy for the BRAF-mutant melanoma therapy.

HLA class I expression predicts prognosis and therapeutic benefits from tyrosine kinase inhibitors in metastatic renal-cell carcinoma patients.

Classical HLA class I antigen is highly involved in antigen presentation and adaptive immune response against tumor. In this study, we explored its predictive value for treatment response and survival in metastatic renal-cell carcinoma (mRCC) patients. A TKI cohort of 111 mRCC patients treated with sunitinib or sorafenib and a non-TKI cohort of 160 mRCC patients treated with interleukin-2 or interferon-α-based immunotherapy at a single institution were retrospectively enrolled. HLA class I expression and cytotoxic T lymphocyte (CTL) density was assessed by immunohistochemistry on tissue microarrays. Association between HLA class I and CTL was also assessed in the TCGA KIRC cohort. In the TKI cohort, down-regulated HLA class I was associated with lower objective response rate of TKI therapy (P=0.004), shorter overall survival (OS) (P=0.001), and shorter progression free survival (PFS) (P<0.001). Multivariate Cox regression model defined HLA expression as an independent prognostic factor for both OS [hazard ratio 1.687 (95% CI 1.045-2.724), P=0.032] and PFS [hazard ratio 2.139 (95% CI 1.376-3.326), P=0.001]. In the non-TKI cohort, HLA class I was not significantly associated with survival. HLA class I expression was associated with CTL infiltration and function, and its prognostic value was more predominant in CTL high-density tumors (P<0.001) rather than CTL low-density tumors (P=0.294). Classical HLA class I expression can serve as a potential predictive biomarker for TKI therapy in mRCC patients. Its predictive value was restricted in CTL high-density tumors. However, further external validations and functional investigations are still required.

Newcastle disease virus mediates pancreatic tumor rejection via NK cell activation and prevents cancer relapse by prompting adaptive immunity.

Pancreatic cancer is the 8th most common cause of cancer-related deaths worldwide and the tumor with the poorest prognosis of all solid malignancies. In 1957, it was discovered that Newcastle disease virus (NDV) has oncolytic properties on tumor cells. To study the oncolytic properties of NDV in pancreatic cancer a single dose was administered intravenously in a syngeneic orthotopic tumor model using two different murine pancreatic adenocarcinoma cell lines (DT6606PDA, Panc02). Tumor growth was monitored and immune response was analyzed. A single treatment with NDV inhibited DT6606PDA tumor growth in mice and prevented recurrence for a period of three months. Tumor infiltration and systemic activation of NK cells, cytotoxic and helper T-cells was enhanced. NDV-induced melting of Panc02 tumors until d7 pi, but they recurred displaying unrestricted tumor growth, low immunogenicity and inhibition of tumor-specific immune response. Arrest of DT6606PDA tumor growth and rejection was mediated by activation of NK cells and a specific antitumor immune response via T-cells. Panc02 tumors rapidly decreased until d7 pi, but henceforth tumors characterized by the ability to perform immune-regulatory functions reappeared. Our results demonstrated that NDV-activated immune cells are able to reject tumors provided that an adaptive antitumor immune response can be initiated. However, activated NK cells that are abundant in Panc02 tumors lead to outgrowth of nonimmunogenic tumor cells with inhibitory properties. Our study emphasizes the importance of an adaptive immune response, which is initiated by NDV to mediate long-term tumor surveillance in addition to direct oncolysis.

Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic Tcell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ Tcells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response tocombination therapy did not appear to be associated with baseline CD8+ Tcell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

Abstract 951: Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity

Abstract Background: Infiltration of cytotoxic T-cells (CTL) in tumors is known to be associated with improved patient’s clinical outcomes. In contrast, local infiltration with regulatory T-cells (Treg) predicts accelerated progression and shorter overall survival. The above observations highlight the need for new measures to promote selective accumulation of CTLs but not Tregs in tumor microenvironments (TME). Here we compare the TME-modulating impact of poly-I:C with a selective TLR3 ligand, rintatolimod showing an induction of CCL22, CXCL12 and expression of COX-2 and downstream suppressive factors, selectively by poly-I:C. Finally, we report on the optimized combinatorial adjuvant to reprogram tumor microenvironment. Materials and Methods: Twelve ovarian cancer specimens were cultured in the presence of IFNα, indometacin (COX-1/2 inhibitor) or/and one of two synthetic TLR3 ligands, poly-I:C (non-selective activator of TLR3 and helicases) or rintatolimod (selective TLR3 ligand). Biopsies were harvested for mRNA measurements and culture supernatants were analyzed for CCL5, CXCL10 and CCL22 concentrations. Alternatively, established ovarian cancer cell lines, monocyte-derived macrophages, THP1 cells or adult fibroblasts were used in analogous experiments. Chemotaxis assays were performed using pre-activated CD8+ T-cells (top chamber) and supernatants from the differentially treated ovarian cancer specimens. Western blot experiments were used to evaluate the impact of NFκB. Blocking experiments using soluble TNFα receptor inhibitor were performed. Results: Both, Poly-I:C and rintatolimod, induced CTL attractant CXCL10. Unexpectedly, poly-I:C but not rintatolimod promoted the expression of the MDSC/Treg attractant CXCL12, which was reversed by addition of indometacin. Furthermore, poly-I:C induced expression of COX-2 and COX-2-dependent suppressive factors (IDO, IL-10). Evaluating the COX-2 cascade, TNFα was found to be increased in the presence of poly-I:C but not rintatolimod. Additionally, Western Blot analysis showed I-κB degradation (as a marker of NF-κB activation), selectively in response to poly-I:C. We observed that the combination of both TLR-3 ligands with IFNα and indomethacin selectively induced the desirable chemokines CCL5 and CXCL10 and suppressed CCL22 in tumor samples with similar results in the macrophages and fibroblasts. The ovarian cancer cell lines showed only minimal expression of chemokines. Conclusion: We demonstrate the feasibility of selective modulation of TME, using different clinically applicable factors and their combinations. We show for the first time, that poly-I:C, but not a selective TLR3 ligand, induces NFκB and undesirable COX2-dependent suppressive factors, which though can be eliminated through addition of a COX-1/2 inhibitor. These effects may be avoided using a selective TLR3 ligand. Citation Format: Marie-Nicole Theodoraki, Ravi Muthuswamy, Jamie Voyten, Francesmary Modugno, Natasa Obermajer, Robert P. Edwards, Pawel Kalinski. Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2017-951

Abstract 1627: Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment

Abstract Hypoxia is a hallmark of cancer and a driver of tumor progression and poor patient outcomes. By generating an immunosuppressive tumor microenvironment that limits cytotoxic T lymphocyte (CTL) infiltration and activation, hypoxia limits the effectiveness of cancer immunotherapy and thus promotes tumor cell evasion of the host immune response. Omniox has developed a first-in-class anti-cancer immunotherapeutic, OMX, specifically designed to reverse tumor hypoxia to enhance cancer immunotherapy efficacy. In preclinical models, we have demonstrated that OMX accumulates in rodent subcutaneous and orthotopic tumors, as well as spontaneous canine melanomas and brain tumors, resulting in significant tumor hypoxia reduction.Here, using multiple subcutaneous syngeneic mouse tumor models (MC38, CT26, 4T1), we assessed OMX effects on intratumoral CTLs and immunosuppressive regulatory T cells (Treg), as well as the anti-tumor potential of OMX as a single agent and in combination with established immunotherapies. Using quantitative immunohistochemistry, we confirmed reports that hypoxic tumor areas are devoid of CTLs. Accordingly, by flow cytometry we observed a negative correlation between tumor hypoxia and CTL infiltration. While OMX single agent treatment did not affect the overall CD45-positive leukocyte population, Treg cells were selectively depleted and the CTL:Treg ratio was substantially increased, suggesting that OMX induced a shift towards immunosensitization. Consistent with this finding, we observed OMX single agent anti-tumor efficacy in MC38 colon tumors. Impressively, anti-tumor effects of OMX single agent were equivalent to that of a single treatment of the checkpoint inhibitor anti-CTLA4. We next assessed whether OMX would enhance the efficacy of checkpoint inhibitors when used in combination. In CT26 colon tumors, OMX exhibited combination anti-tumor activity with anti-CTLA4, giving rise to faster cures and a greater number of complete and durable responders compared to anti-CTLA4 alone. Of note, this enhanced response was observed for both early-stage and late-stage CT26 tumors. In 4T1 breast tumors, known to be insensitive to checkpoint inhibitors, treatment of early-stage (~60mm3) tumors with combination OMX and anti-PD1 resulted in a 27% response rate, compared to a 0% response rate to anti-PD1 alone. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic tumor areas, induces a microenvironmental change from an immunosuppressive to an immunopermissive state. Given that OMX is well-tolerated in both small and large animals, and that its mechanism of action is upstream of numerous major immunosuppressive pathways, OMX holds great clinical potential to synergize with multiple immunotherapeutic agents to enhance tumor control by restoring anti-cancer immune responses in cancer patients. Citation Format: Kevin G. Leong, Yuqiong Pan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2017-1627

Abstract SY06-03: Reprogramming the tumor microenvironment to improve responses to therapy

Abstract Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells, and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists, leading to a near tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and thus renders tumors responsive to immunotherapy. To test this hypothesis in human PDAC patients, we have opened a phase I clinical trial testing the safety and impact on the tumor microenvironment of the FAK inhibitor defactinib, in combination with pembrolizumab and gemcitabine (NCT02546531). These studies are ongoing in patients with advanced cancer, including metastatic PDAC. Early biomarker studies on PDAC tissue collected pre- and post-therapy suggest that this combination impacts the immunosuppressive TME. Citation Format: David G. Denardo. Reprogramming the tumor microenvironment to improve responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY06-03. doi:10.1158/1538-7445.AM2017-SY06-03

PRKCI promotes immune suppression in ovarian cancer.

A key feature of high-grade serous ovarian carcinoma (HGSOC) is frequent amplification of the 3q26 locus harboring PRKC-ι (PRKCI). Here, we show that PRKCI is also expressed in early fallopian tube lesions, called serous tubal intraepithelial carcinoma. Transgenic mouse studies establish PRKCI as an ovarian cancer-specific oncogene. Mechanistically, we show that the oncogenic activity of PRKCI relates in part to the up-regulation of TNFα to promote an immune-suppressive tumor microenvironment characterized by an abundance of myeloid-derived suppressor cells and inhibition of cytotoxic T-cell infiltration. Furthermore, system-level and functional analyses identify YAP1 as a downstream effector in tumor progression. In human ovarian cancers, high PRKCI expression also correlates with high expression of TNFα and YAP1 and low infiltration of cytotoxic T cells. The PRKCI-YAP1 regulation of the tumor immunity provides a therapeutic strategy for highly lethal ovarian cancer.

A TLR3-Specific Adjuvant Relieves Innate Resistance to PD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy

Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF)3-IFN-β axis in DCs exclusively participated in CD8+ Tcell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human exvivo studies disclosed that ARNAX+antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.

Immune Cell Infiltrates in Hippocampal Sclerosis: Correlation With Neuronal Loss.

Immune mechanisms have been increasingly recognized in the pathogenesis of hippocampal sclerosis (HS), but infiltration of cytotoxic T-cells and its pathological significance in patients with HS has not been explored. We examined 30 cases of surgically resected hippocampi, including 16 International League Against Epilepsy (ILAE) type 1, 9 ILAE type 2, 1 ILAE type 3 HS, and 4 ILAE No-HS, as well as 6 autopsy No-HS hippocampi. The HS hippocampi showed sparse to scattered CD8-positive T-cells, rare CD4-positive T-cells, and a modest increase in CD68-positive microglia/macrophages, which were significantly more numerous than those in the No-HS controls. The infiltration of CD8-positive T-cells was significantly greater in the CA1 subfield than other subfields of type 1 and type 2 HS. The numbers of CD8-positive T-cells positively correlated with those of CD4-positive T-cells; there was a lower ratio of CD4/CD8-positive T-cells. There were positive correlations between these cells and scores of neuronal loss but no significant correlation between the infiltration of these cells and epilepsy disease duration or age of epilepsy onset. These findings suggest that an autoimmune process may be involved in the pathogenesis of HS and infiltration of immune cells, particularly CD8-positive cytotoxic T-cells, may contribute to neuronal loss in HS.

Identification of distinct subgroups of EBV-positive post-transplant diffuse large B-cell lymphoma

Post-transplantation lymphoproliferative disorder is an aggressive complication of transplantation, most frequently of diffuse large B-cell lymphoma morphology and associated with Epstein–Barr virus (EBV) infection/reactivation. In this study the microenvironment of EBV+ (n=23) and EBV− (n=9) post-transplant non-germinal center B-cell diffuse large B-cell lymphoma was characterized. Of EBV+ cases somatic hypermutation analysis, gene expression profiling, and extensive phenotyping were performed. Our results demonstrated variable cytotoxic T-cell infiltration and significantly increased CD163+ M2 macrophage infiltration in EBV+ compared with EBV− post-transplant diffuse large B-cell lymphoma. On the basis of IgM staining and hypermutation analysis, two EBV+ post-transplant diffuse large B-cell lymphoma subgroups were identified: IgM+ tumors lacking somatic hypermutations and IgM− tumors harboring somatic hypermutations. IgM− tumors arose late following transplantation (median interval: 16 months), mainly in kidney recipients. IgM+ tumors on the other hand arose early (median interval: 3 months, P-value=0.0032), almost exclusively following stem cell transplantation and were associated with worse outcome (median survival 1 month for IgM+versus 41 months for IgM− tumors, log-rank/Wilcoxon P-value 0.07/0.04). Notably, IgM+ tumors were characterized by plasma cell features (monotypic kappa/lambda expression, high MUM1 expression, and partial CD138 expression) and a high proliferation index. Consistent with the plasma cell phenotype, unfolded protein response signaling was upregulated. In contrast, IgM− EBV+ post-transplant diffuse large B-cell lymphoma did not express kappa, lambda, IgD, or CD138 and expressed limited MUM1. In these tumors T-cell signaling was enhanced associated with increased T-cell infiltration compared with IgM+ cases. Overall, our results allow further molecular classification of EBV+ post-transplant diffuse large B-cell lymphoma and provide a rationale for the use of subtype-specific-targeted therapies (eg, bortezomib in IgM+ tumors). Our findings also provide a biological basis for the clinical differences between post-transplant lymphoproliferative disorder following solid organ and stem cell transplantation, which are regarded as different disorders.