Xiaoyu Pan

Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy

Purpose: Immunomodulatory drugs differ in mechanism-of-action from directly cytotoxic cancer therapies. Identifying factors predicting clinical response could guide patient selection and therapeutic optimization. Experimental Design: Patients (N = 41) with melanoma, non–small cell lung carcinoma (NSCLC), renal cell carcinoma (RCC), colorectal carcinoma, or castration-resistant prostate cancer were treated on an early-phase trial of anti–PD-1 (nivolumab) at one institution and had evaluable pretreatment tumor specimens. Immunoarchitectural features, including PD-1, PD-L1, and PD-L2 expression, patterns of immune cell infiltration, and lymphocyte subpopulations, were assessed for interrelationships and potential correlations with clinical outcomes. Results: Membranous (cell surface) PD-L1 expression by tumor cells and immune infiltrates varied significantly by tumor type and was most abundant in melanoma, NSCLC, and RCC. In the overall cohort, PD-L1 expression was geographically associated with infiltrating immune cells (P < 0.001), although lymphocyte-rich regions were not always associated with PD-L1 expression. Expression of PD-L1 by tumor cells and immune infiltrates was significantly associated with expression of PD-1 on lymphocytes. PD-L2, the second ligand for PD-1, was associated with PD-L1 expression. Tumor cell PD-L1 expression correlated with objective response to anti–PD-1 therapy, when analyzing either the specimen obtained closest to therapy or the highest scoring sample among multiple biopsies from individual patients. These correlations were stronger than borderline associations of PD-1 expression or the presence of intratumoral immune cell infiltrates with response. Conclusions: Tumor PD-L1 expression reflects an immune-active microenvironment and, while associated other immunosuppressive molecules, including PD-1 and PD-L2, is the single factor most closely correlated with response to anti–PD-1 blockade. Clin Cancer Res; 20(19); 5064–74. ©2014 AACR.

Cutting Edge: Accelerated Autoimmune Diabetes in the Absence of LAG-3

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4+ and CD8+ T cell homeostasis. Lag3−/− NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4+ T cells and, to a lesser extent, CD8+ T cells. Lag3−/− mice exhibited accelerated, invasive insulitis, corresponding to increased CD4+ and CD8+ T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4+ T cells and islet specific glucose-6-phosphatase-specific CD8+ T cells were significantly increased in the islets of Lag3−/− mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4+ and CD8+ T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

Tumor-Associated Antigen Expressing Listeria monocytogenes Induces Effective Primary and Memory T-Cell Responses Against Hepatic Colorectal Cancer Metastases

PurposeDespite advances in therapy for the treatment of metastatic colorectal cancer, many patients die of hepatic disease. Current immunotherapeutic strategies are likely limited by inhibitory signals from the tumor. To successfully eliminate tumor deposits within an organ, an appropriate immunologic milieu to amplify antitumor responses must be developed.MethodsWe used a murine model utilizing the CT26 colon cancer cell line to analyze primary and memory tumor-specific T-cell responses induced by an attenuated actin A and internalin B deleted immunodominant tumor-associated antigen expressing strain of Listeria monocytogenes for the treatment of metastatic colorectal cancer.ResultsTreatment of mice bearing established hepatic metastases with this L. monocytogenes strain led to the generation of a strong initial tumor-specific cytotoxic CD8+ T-cell response that successfully treated 90% of animals. Tumor antigen-specific central and effector memory T cells were also generated and protected against tumor rechallenge. These cell populations, when measured before and after tumor rechallenge, showed a marked expansion of antigen-specific effector CD8+ effector memory T cells. This strain of L. monocytogenes was able to down-modulate the expression of the immune checkpoint molecule, PD-1, within the tumor microenvironment but had variable effects on CTLA-4 expression.ConclusionsThis L. monocytogenes strain generated a highly effective antitumor T-cell response, providing a basis for the development of this vaccine platform in patients with liver metastases.

Glycolipid antigens for treating hepatic colorectal cancer metastases and their effect on the therapeutic efficacy of live attenuated Listeria monocytogenes.

Previous work demonstrated that a subset of natural killer T cells in mice decreased the antitumor efficacy of live attenuated Listeria monocytogenes where the actin A and internalin B genes were genetically deleted (LMD) against murine hepatic colorectal cancer metastases. Therefore, we hypothesized that the use of specific glycolipids known to selectively stimulate natural killer T-cell subsets used alone or co-administered with LMD would increase survival. We found that early or multiple administrations of glycolipids after tumor challenge had a strong impact on survival with or without LMD. Solitary administration or treatment given later was less efficacious but still showed a strong trend toward enhancing the antitumor activity of LMD. These results underscore the potential of glycolipids in the treatment of hepatic metastases and encourage further investigations into the immunomodulation of natural killer T cells to enhance the antitumor activity of LMD.

Abstract 581: Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG

Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice. Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response. Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice. Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer. Citation Format: Ofer Levy, Chris Chan, Gady Cojocaru, Spencer Liang, Eran Ophir, Sudipto Ganguly, Maya Kotturi, Tal Friedman, Benjamin Murter, Liat Dassa, Ling Leung, Shirley Greenwald, Meir Azulay, Sandeep Kumar, Zoya Alteber, Xiaoyu Pan, Andy Drake, Ran Salomon, Arthur Machlenkin, John Hunter, Zurit Levine, Drew Pardoll, Mark White. Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG [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 581. doi:10.1158/1538-7445.AM2017-581

Abstract 5033: Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ

Epigenetic treatment and immune checkpoint blockade are novel therapeutic approaches being investigated in NSCLC. Our previous studies showed that DNA methyltransferase inhibitor azacitidine (AZA) treatment in NSCLC cell lines led to up-regulation of genes related to the response to interferon and adaptive immune attack. We hypothesize that Interferon-γ(IFN-γ), an immune effecter made primarily by activated T cells and NK cells, has a direct tumor suppression effect in NSCLC cells, which can be enhanced by the DNA hypomethylation agent. To test this hypothesis, NSCLC cells (H838 and/or H1299) were first treated with or without 500nM AZA for 3 days, and then received IFN-γ. The samples were subsequently tested for cell viability, apoptosis, cell cycle, and gene expression assays. This preclinical study is part of the collective efforts to investigate the efficacy of combined epigenetic and immune therapy in NSCLC. IFN-γ causes moderate direct growth inhibition in H838 and H1299 cells, which was enhanced, using cell viability assays as a readout, by pretreatment with AZA. The AZA sensitizing effect lasted at least 3 weeks and was not detectable 8 weeks after stopping treatment. This timing is consistent with initial reprogramming of the cells and eventual waning of this effect. Flow cytometry studies showed that AZA enhances lung cell apoptosis induced by IFN-γ. However, no significant change in cell cycle was observed. To examine the mechanisms behind these effects on cell growth, we further determined how AZA altered IFN-γ induced gene expression in H1299 cells, first using Agilent expression arrays, and then validating specific changes in key IFN and cell death pathway genes with RT-PCR based methods. IFN-γ significantly up-regulated a group of known IFN targeted genes, including IFI27, IFITM1, ISG20, ICAM1, CCL5, CXCL10, MX1 and others, and this upregulation was further enhanced by AZA pretreatment. AZA also enhanced IFN-γ induced expression of CASP1, CASP4, TNFSF 10, and BCL2A. These AZA enhanced IFN-γ induced genes are important in tumor immune response and evasion, cell death, cytokine response and other critical cellular process. Our studies demonstrated that AZA enhanced lung cancer cell response to the immune effecter IFN-γ. This supports the clinical exploration of epigenetic “priming” and its combination with subsequent immune therapy in NSCLC treatment. It may also provide mechanistically derived biomarkers that can be used to monitor and predict epigenetic and immune response in NSCLC. Citation Format: Kai He, Xin Zhang, Ludmila Danilova, Xiaoyu Pan, Julie Brahmer, Drew Pardoll, Malcolm Brock, Stephen Baylin, James Herman, John Wrangle. Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2015-5033

Abstract 5031: Effects of BRAF and MEK inhibitors, dabrafenib and trametinib, on the immune system and in combination with immunomodulatory antibodies targeting PD1, PD-L1 and CTLA-4

The immunological effects of dabrafenib and trametinib and whether they potentiate or antagonize the activity of immunomodulatory antibodies are not well understood. We assessed the immunological effects of dabrafenib and trametinib at clinically relevant exposure concentrations on both immune and tumor cells in vitro and in vivo, and tested their anti-tumor efficacy in combination with immunomodulatory antibodies in immune-competent syngeneic mouse models. Human CD4+ and CD8+ T cells isolated from healthy volunteers were treated with trametinib and dabrafenib either alone or in combination, and with or without anti-CD3/anti-CD28 bead activation (concurrently or sequentially). Dabrafenib alone enhanced pERK expression levels with no changes of pAKT and pS6 proteins, and had no suppressive impact on human CD4+ or CD8+ T cell proliferation, apoptosis and cytokine production in response to T cell activation. Trametinib alone reduced the pERK levels with no changes in pAKT and apoptosis. However trametinib resulted in partial inhibitory effects on T cell proliferation, pS6 proteins and cytokine expression. These inhibitory effects were transient and only observed if cells were treated with trametinib prior to or simultaneously with T cell activation, while trametinib had little or no suppressive effects on activated T cells. Adding dabrafenib partially offset the transient inhibitory effects caused by trametinib alone. Similarly, gene expression profiling showed that trametinib partially decreased the expression levels of a subset of cytokines and chemokines (e.g. IL1, IL2, IL8, IL10, TNFa, CCL2) and activation/regulation markers (e.g. CD69, CD25, PD1, CTLA4) when trametinib was added prior to or simultaneously with T cell activators. Multi-color flow cytometry confirmed cell surface changes in the expression of CD69, CD25, PD1, OX40 and CTLA4. However, the expression levels of CD69 and OX40 were still well above non-activated T cells. On tumor cells, dabrafenib and trametinib up-regulated HLA molecules and melanoma antigen MART1 expression, and down regulated immune-suppressive factors such as PD-L1, VEGF and IL8 etc in BRAFV600E melanoma cells. Combinations of trametinib with immunomodulators targeting PD1, PD-L1 or CTLA4 in murine syngeneic tumor models are underway and will be presented at the meeting. These findings to date support clinical exploration of dabrafenib and/or trametinib in combination with specific immunomodulatory antibodies. Citation Format: Li Liu, Patrick Mayes, Stephen Eastman, Hong Shi, Sapna Yadavilli, Xiaoyu Pan, Jingsong Yang, Laura Seestaller-Wehr, Shu-Yun Zhang, Chris Hopson, Lyuben Tsvetkov, Junping Jing, James Smothers, Drew M. Pardoll, Axel Hoos. Effects of BRAF and MEK inhibitors, dabrafenib and trametinib, on the immune system and in combination with immunomodulatory antibodies targeting PD1, PD-L1 and CTLA-4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5031. doi:10.1158/1538-7445.AM2014-5031

Abstract 4619: Epigenetic therapy and sensitization of lung cancer to immunotherapy.

Epigenetic alterations driving carcinogenesis and cancer progression can be specifically targeted by the demethylating agent azacitidine (Aza) and the histone deacetylase inhibitor entinostat. While this treatment combination has been effective in a limited number of patients (pts) with treatment-refractory non-small cell lung cancer (NSCLC), we observed clinical benefit in 5 of 5 patients who received immunotherapy with PD-1/PD-L1 pathway blockade immediately following epigenetic therapy. Three of 5 pts developed partial tumor regressions (RECIST criteria, duration 10+ to 20+ mo.) and 2 pts had stable disease ≥6 mo. This compares to the objective response + SD rates of NSCLC to monotherapy with anti-PD-1 (18% + 5%) or anti-PD-L1 (10% + 12%). To understand how epigenetic therapy may synergize with blockade of the immunosuppressive PD-1 pathway, we used genome wide methylation and expression profiling on 8 NSCLC cell lines treated with low dose Aza. We discovered complex immunomodulatory effects of Aza with up-regulation of diverse immune related pathways including Jun/Jnk, NFKB, viral defense, type I interferon signaling, the inflammasome, antigen processing and presentation and immune evasion including up-regulation of PD-L1 expression. Multiple cancer-testes antigens were also up-regulated, thereby conferring de novo antigenicity. Supporting the idea that Aza acts specifically through inhibition and degradation of DNA methyltransferase proteins, colon cancer cells genetically haplo-insufficient for DNMT1 and devoid of DNMT3b mirror the immunomodulatory effects of Aza. Upstream events potentially controlling these pathways were defined, and prominent among them was up-regulation of the transcription factor, interferon regulatory factor 7 (IRF7), a DNA hypermethylated gene. These data were used to query hundreds of primary NSCLC samples from the Cancer Genome Atlas project (TCGA). A low basal expression signature of interferon pathway related genes was significantly associated with low IRF7 expression and promoter methylation in squamous tumors. Another hypermethylated transcription factor, PITX1, which inhibits a subset of type I interferon signaling genes, tracked with non-squamous cancers. Together, these findings support a model in which epigenetic modulation activates innate and adaptive immune responses within the tumor microenvironment together with induction of counter-regulatory immune checkpoint ligands which can be therapeutically blocked with antibodies. Based on these findings, a clinical trial testing the efficacy of DNMT and HDAC inhibition combined with PD-1 pathway blockade is under development. This work will form the basis for an immune-classification of NSCLC, as well as biomarker discovery for a novel therapeutic paradigm combining epigenetic and immunotherapy with potentially synergistic activity against the world9s most deadly malignancy. Supported by Stand Up to Cancer. Citation Format: John Wrangle, Wei Wang, Alexander Koch, Hariharan Easwaran, Helai Mohammad, Princy Parsana, Frank Vendetti, Kristen Rodgers, Xiaoyu Pan, Kirsten Harbom, Cynthia Zahnow, Janis Taube, Julie Brahmer, Peter Jones, Suzanne Topalian, Charles Rudin, Malcolm Brock, Drew Pardoll, Stephen Baylin. Epigenetic therapy and sensitization of lung cancer to immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2013-4619

Abstract LB-456: The intraductal injection of 5-fluorouracil induces immune response to eradicate and prevent breast cancer

Chemotherapy has been shown to act as an immune response modifier. Many groups, incuding our own, are using chemotherapy to induce immune responses such as by using low doses of cyclophosphamide. Our previous work has demonstrated that anti-cancer agents can eliminate new/existing tumors with minimal toxicity in rat and mouse models of breast cancer. Here we demonstrate that intraductal (i.duc), but not the intravenous (IV) route of injection, of chemotherapy to mammary glands alters the tumor environment to effectively induce immune effector cells. We used the mouse HER2/neu (neu/N) transgenic, spontaneous mammary tumor model. 5-fluorouracil (5FU) was administered intraductally to the mammary glands on the left side of parous mice whereas the mammary glands on right side received no treatment (NT). The mammary tumor incidence in the 5FU treated side was significantly lower compared to mice that received NT, and IV treated group. Interestingly the incidence of mammary tumors in the untreated side of 5FU-treated mice was also significantly lower compared to the NT and IV group. We hypothesized that the protection afforded to the contralateral chain of mammary glands by ipsilateral i.duc administration of 5FU, may be mediated through an immunological mechanism. Twenty week parous mice were administered 5FU either through i.duc injection, only to left side teats, IV injection, or NT. The mice received 5FU 2 times in a 4 week interval. A week after the second treatment, the mice were sacrificed and the regional lymphnodes (RLN) of the 3 rd and 4 th mammary glands and spleen were removed. We isolated lymphocyte from RLN and spleen to analyze by flowcytometry. The number of CD8 T cell showed no change among the groups in the RLN but was significantly lower in the i.duc group in the spleen. The number of CD62L LOW+ T cell in 5FU treated side of RLN was significantly higher compared to the IV and NT group. On the other hand the number of CD62L LOW+ was low in the spleen. To study the population of systemic memory T cell in the peripheral blood, blood was harvested into Trucount tubes and analyzed with memory T cells surface marker. Twenty week parous mice was administered 5FU i.duc injection only to the left side, IV injection, or NT every 4weeks for a total of 3 times. Cancer cells were injected into the left side of 4 th mammary gland fat pad of the mice at 16 weeks after initial procedure. One hundred μl blood was collected 5 days before and 5 days after cancer cell injection. The mice treated by i.duc 5FU showed increased numbers of CD95 + /CD62L low effector memory T (T EM ) cells, whereas the mice treated with IV 5FU did not recruit T EM cells significantly. There was no significant change in CD95 + /CD62L high central memory T cells before and after treatment and among the groups. In summary, i.duc administration of 5FU into mammary glands effectively induced immune effector cells and prevented mammary tumor growth in neu/N transgenic mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-456. doi:10.1158/1538-7445.AM2011-LB-456

Abstract 2935: Modulation of immune checkpoint B7-H1/B7-DC/PD-1 interaction in combination with live attenuated Listeria monocytogenes expressing a tumor associated antigen effectively treats hepatic colorectal cancer metastases

Introduction: The successful eradication of tumor deposits by immunotherapy requires the generation of an appropriate primary and memory immune effector immune response as well as the establishment of a tumor micro-environment within that organ to target, traffic and amplify the appropriate immune response. B7-H1 is expressed on dendritic cells (DCs), macrophages, T/B cells and on various cancer cell lines. B7-H1 when bound to its T-cell receptor, programmed death-1 (PD-1) induces a negative regulatory signal inhibiting T cell responses. These are particularly attractive targets of therapy in metastatic colorectal cancer as the cancer and the liver microenvironment express B7H1, while tumor infiltrating CD8+ T cells express PD-1. It appears that cancer is able to exploit the B7H1-PD-1 inhibitory mechanism thus making the blockade of this interaction capable of enhancing our already validated vaccine platform of doubly attenuated Listeria Monocytogenes (LM). Methods and Results: Isolated hepatic metastases were generated in Balb/c mice and treated with intraperitoneal injections of 0.1 x LD 50 of LM on postoperative days 3, 6, 9. In addition, intravenous injection of a mouse anti-mouse B7-H1 blocking antibody was given on days 4,7,10 and compared to mice given vaccine alone, antibody alone, and no treatment. We analyzed immune cell populations in the liver with flow cytometry to define the activity, specificity and kinetics. After blockade of B7-H1 we performed survival analysis and in vitro studies for T cell proliferation of activated T cells. Results: Mice treated with LM and B7-H1 showed a 60% survival, LM alone showed 30% survival, while antibody alone and untreated mice did not survive (Figure 1). We first demonstrated B7-H1 expression on colon cancer cell line CT26. We then found CD8 + T cells and conventional dendritic cells (cDC) treated with LM, showed an up-regulation of B7-H1. However, these receptors upregulation were both abgrogated when combined with B7H1 blocking antibody. When B7H1 blocking antibody was used alone, there was a decrease in B7H1 expression but an increased expression on PD-1 on CD8+ T cells. T cell proliferation assay in vitro showed sustained T cell activity with B7-H1 blockade even in the presence of tumor. Conclusion Blockade of B7-H1 is an effective method of overcoming immune evasion and improves the efficacy of LM vaccine in the treatment of metastatic colorectal cancer to the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2935.