Durable Antitumor Immunity Research Articles

Cryo-thermal therapy induces macrophage polarization for durable anti-tumor immunity

Many cancer therapies are being developed for the induction of durable anti-tumor immunity, especially for malignant tumors. The activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), can bridge innate and adaptive immune responses against tumors. However, APCs have an immunosuppressive phenotype and reversing it for effective tumor-specific antigen presenting is critical in developing new cancer treatment strategies. We previously developed a novel cryo-thermal therapy to treat malignant melanoma in a mouse model; long-term survival and durable anti-tumor immunity were achieved, but the mechanism involved was unclear. This study revealed cryo-thermal therapy-induced macrophage polarization to the M1 phenotype and modulated the phenotypic and functional maturation of DCs with high expression of co-stimulatory molecules, increased pro-inflammatory cytokine production, and downregulated immuno-inhibitory molecule expression. Further, we observed CD4+ T-cell differentiation into Th1 and cytotoxic T-cell sub-lineages and generation of cytotoxic CD8+ T cells, in which M1 macrophage polarization had a direct, important role. The results indicated that cryo-thermal-induced macrophage polarization to the M1 phenotype was essential to mediate durable anti-tumor immunity, leading to long-term survival. Thus, cryo-thermal therapy is a promising strategy to reshape host immunosuppression, trigger persistent memory immunity for tumor eradication, and inhibit metastasis in the long term.

Abstract B137: Preclinical evaluation of a Wilms’ tumor protein 1-targeted interleukin-15 dendritic cell vaccine: T-cell activity and batch production

Abstract Encouraging results from clinical trials demonstrate that dendritic cell (DC) vaccination is a valuable tool in cancer immunotherapy. Empowering DC vaccine immunogenicity to improve clinical outcome is at center stage in the rapidly evolving immunotherapeutic landscape. Developing a unique type of DC vaccine, so-called interleukin (IL)-15 DCs generated with IL-15 and strong immune stimulating maturation signals, we could demonstrate superior in vitro activities at both adaptive as well as innate immunity as compared to gold-standard IL-4 DCs. In this light, IL-15 DCs are capable of activating NK cells and gamma-delta T-cells, whereas IL-4 DCs are not. Designed to induce durable antitumor T-cell immunity, this pre-clinical research focuses on the improvement of Wilms’ Tumor protein 1 (WT1)-targeted T-cell activity. The WT1 antigen is overexpressed in a wide variety of human cancers and thus appears to fulfill the criteria of a universal tumor-associated antigen. The capacity of our novel IL-15 DCs to induce WT1-specific immunity is assessed using two in-house developed tumor antigen-specific T-cell assays. The first series of experiments demonstrate in a head-to-head comparison that WT1 mRNA-electroporated IL-15 DCs perform at least as well as IL-4 DCs in triggering a WT1 T-cell receptor (TCR)-transfected T-cell line. Using an autologous primary TCR-engineered CD8+ T-cell approach, we are now comparing their WT1-specific antitumor function. With its unique characteristics, including IL-15-transpresentation and interferon-gamma secretion, IL-15 DCs are expected to significantly promote WT1-specific T-cell-mediated tumor cell killing. Enabling its clinical application, we also evaluated the upscaled IL 15 DC vaccine production and cryopreservation processes. We were able to employ the three-day culture protocol in clinically suitable culture flasks. In addition, the use of an optimized cryopreservation medium results in a pre- to-post cryopreservation yield of 81.3 ± 7.4 % with a preserved phenotype and functionality. High-scale production and cryopreservation allows for the implementation of DC vaccination in multimodal treatment schedules. Underscoring the tumor antigen-specific T-cell-stimulating capacity among previously described superior characteristics and warranting batch production of patient-specific DC vaccines, further strengthens the impetus to bring WT1-loaded IL-15 DCs to the clinic. Citation Format: Maarten Versteven, David Damoiseaux, Diana Campillo-Davo, Heleen Van Acker, Hans De Reu, Sébastien Anguille, Zwi N Berneman, Evelien L Smits, Viggo F Van Tendeloo, Eva Lion. Preclinical evaluation of a Wilms’ tumor protein 1-targeted interleukin-15 dendritic cell vaccine: T-cell activity and batch production [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B137.

Magnitude of Therapeutic STING Activation Determines CD8+ T Cell-Mediated Anti-tumor Immunity

Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a Tcell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector Tcells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific Tcell activation, and in the context of optimized Tcell responses, TNFα is dispensablefor tumor control. In a poorly immunogenic model,S100 combined with CPIs generates asurvivalbenefit and durable protection. These resultsprovidefundamental mechanistic insights into STING-induced anti-tumor immunity.

Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity

ABSTRACTMetastatic melanoma is a significant clinical problem with a 5-year survival rate of only 15–20%. Recent approval of new immunotherapies and targeted inhibitors have provided much needed options for these patients, in some cases promoting dramatic disease regressions. In particular, antibody-based therapies that block the PD-1/PD-L1 checkpoint inhibitory pathway have achieved an increased overall response rate in metastatic melanoma, yet durable response rates are reported only around 15%. To improve the overall and durable response rates for advanced-stage melanoma, combined targeted and immune-based therapies are under investigation. Here, we investigated how the natural products called schweinfurthins, which have selective anti-proliferative activity against many cancer types, impact anti-(α)PD-1-mediated immunotherapy of murine melanomas. Two different compounds efficiently reduced the growth of human and murine melanoma cells in vitro and induced plasma membrane surface localization of the ER-resident protein calreticulin in B16.F10 melanoma cells, an indicator of immunogenic cell death. In addition, both compounds improved αPD-1-mediated immunotherapy of established tumors in immunocompetent C57BL/6 mice either by delaying tumor progression or resulting in complete tumor regression. Improved immunotherapy was accomplished following only a 5-day course of schweinfurthin, which was associated with initial tumor regression even in the absence of αPD-1. Schweinfurthin-induced tumor regression required an intact immune system as tumors were unaffected in NOD scid gamma (NSG) mice. These results indicate that schweinfurthins improve αPD-1 therapy, leading to enhanced and durable anti-tumor immunity and support the translation of this novel approach to further improve response rates for metastatic melanoma.

Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity

Immune stimulation has emerged as a promising approach to the treatment of neoplastic diseases. Currently approved therapeutics, such as anti-CTLA4 and anti-PD1, are primarily aimed at blocking inhibitory signaling by immune cells. An alternative and potentially synergistic approach would involve activation of immune pathways by agonism of stimulatory receptors, such as CD40. Agonistic antibodies, while promising in principle, have encountered significant barriers in clinical trials limited by the systemic toxicity of such approaches. Using a mouse model humanized for both Fc receptors and CD40, we previously demonstrated enhanced antitumor activity with an Fc-modified antibody. We now demonstrate that this model recapitulates the platelet and hepatic toxicities seen with anti-CD40 antibodies in patients, providing a predictive measure of the dose-limiting activity of this approach. We further show that such toxicity can be circumvented and durable systemic antitumor immunity achieved by intratumoral delivery of an Fc-engineered anti-CD40 agonistic antibody.

Metastatic Melanoma: Recent Therapeutic Progress and Future Perspectives.

The prognosis of patients with metastatic melanoma has dramatically improved in recent years with the introduction of two new therapeutic strategies. BRAF and MEK inhibitors are small molecules that are able to block the mitogen-activated protein kinase (MAPK) pathway, which is constitutively activated by recurrent BRAF V600 mutations in 45% of melanoma patients. These agents were shown to provide a rapid and strong response but are often limited by a high rate of secondary resistance. Monoclonal antibodies against the immune checkpoints cytotoxic T lymphocyte-associated antigen4 (CTLA-4) and programmed death1 (PD-1) can restore an efficient and durable anti-tumor immunity, even following treatment discontinuation. Anti-PD-1 antibodies were shown to prolong survival of metastatic melanoma patients and a real cure seems to be obtainable in some patients. Many more therapies are currently under investigation, given that 50% of patients still do not have long-term benefits from approved treatments. The main goal is to avoid or circumvent primary or secondary immune resistance to anti-PD-1 therapy not only by targeting other players in the tumor microenvironment but also by optimizing treatment sequencing and combining anti-PD-1 with other treatments, especially with BRAF and MEK inhibitors. The unexpected major successes of immunotherapies in melanoma have opened the way for the development of these treatments in other cancers. In this review, we describe the different available treatments, their toxicities, and the key components of our decisional algorithms, and give an overview of what we expect to be the near future of melanoma treatment.

Abstract 631: Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors

Abstract Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) induces stable tumor regression in preclinical models, yet the underlying immune correlates are not fully understood. ADU-S100, a CDN under clinical evaluation, was administered IT with an optimized dosing regimen to explore the immune requirements for antitumor efficacy in mouse syngeneic tumor models. We show that CD8+ T cells are necessary and sufficient for durable antitumor immunity elicited by ADU-S100 and that activation of STING in hematopoietic cells mediates CD8+ T cell induction. Both type I IFN and TNFα, which are induced by STING pathway activation, influence the antitumor immune response. The combination of ADU-S100 and anti-PD1 treatment enhances CD8+ T cell-dependent, noninjected tumor control that correlates with an enhanced effector profile of CD8+ T cells in the tumor. Combination of ADU-S100 with checkpoint inhibition also enhances durable immunity in a poorly immunogenic tumor model. Together, these results elucidate the immune correlates to STING-mediated antitumor efficacy and highlight the potential of combining STING agonists with checkpoint inhibition in the clinic. Citation Format: Anthony L. Desbien, Kelsey Sivick Gauthier, Leticia Corrales, Gabrielle Reiner, Laura Hix Glickman, George Katibah, Thomas E. Hudson, Uyen Vu, Natalie H. Surh, Brian Francica, Weiwen Deng, David B. Kanne, Justin J. Leong, Chudi Ndubaku, Ken Metchette, Jeffery M. McKenna, Steven L. Bender, Meredith L. Leong, Thomas W. Dubensky Jr., Andrea van Elsas, Sarah M. McWhirter. Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors [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 631.

Toca 6: A phase 1b study of Toca 511 and Toca FC in patients with advanced solid tumors or lymphoma.

TPS2613 Background: Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector that selectively infects cancer cells. Toca 511 spreads through tumors, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug, Toca FC (investigational, extended-release 5-FC), into 5-FU within the tumor microenvironment. Preclinical studies show 5-FU kills infected dividing cancer cells and diffuses and kills surrounding cancer cells, myeloid derived suppressor cells, and tumor associated macrophages, thus reestablishing tumor immunity. A prior clinical study showed CD protein expression in resected high grade glioma tumors after intravenous (IV) Toca 511 (1Cloughesy T, Walbert T, Bota D, et al. Neuro Oncol 2016; 18(suppl 6):vi17). In animal models of metastatic colorectal cancer, IV Toca 511 infected metastases; subsequent 5-FC treatment resulted in decreased tumor size, improved survival, and durable antitumor immunity (2Yagiz K, Rodriguez-Aguirre ME, Espinoza FL, et al. Molecular Therapy: Oncolytics. 2018; 8:14-26). Methods: Toca 6 is a Phase 1b, multicenter, open-label study (NCT02576665) investigating changes in immune activity after treatment with Toca 511 & Toca FC in patients with advanced solid tumors or lymphoma. Toca 511 is injected IV daily for 3 days, then intratumorally following biopsy or, for patients with brain metastases, into resection cavity walls following resection. Oral Toca FC is started ~4 weeks later and repeated every 4-6 weeks. Changes from baseline in intratumoral immune activity (infiltrating T-cell subpopulations, B cells, monocytes) at 4 weeks after start of Toca FC are assessed. Peripheral blood is analyzed for effector, memory, Treg, and myeloid lineage cells. Viral RNA, DNA, and CD protein expression in tumor after IV Toca 511 are measured. Safety and efficacy are assessed. Approximately 30 patients will be enrolled at 4 sites in the United States. Enrollment progress by tumor type will be provided at the time of the meeting. Clinical trial information: NCT02576665.

Therapeutic oncolytic viruses: clinical advances and future directions

The present review will highlight recent advances in the clinical application of oncolytic viral therapy. Until recently, oncolytic viral researchers saw the immune system as an enemy that would clear the virus from the bloodstream. However, researchers now understand that sustained responses are seen in those patients with more robust antitumor immune responses. Much of the current focus in oncolytic viral research is trained on manipulation of the immune system to affect cancer cell killing in the tumor microenvironment and to facilitate durable systemic antitumor immunity. Many investigators have demonstrated synergistic effects of checkpoint inhibition and other immune therapies with viral administration. At the same time, insertion of various markers enables noninvasive deep tissue imaging. Finally, following regulatory approval in the United States and Europe, unbridled clinical use of T-VEC for patients with metastatic melanoma is also generating large volumes of patient data that will help elucidate strengths and weaknesses of oncolytic viral therapy. Perhaps the most telling sign of the field's future is a seismic shift in clinical trials with more investigators combining virus and immunotherapies. This article reviews the current state of therapeutic oncolytic viruses in clinical use, and explores future directions of the field.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma.

Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Depletion of Tumor-Associated Macrophages with a CSF-1R Kinase Inhibitor Enhances Antitumor Immunity and Survival Induced by DC Immunotherapy.

New immunotherapeutic strategies are needed to induce effective antitumor immunity in all cancer patients. Malignant mesothelioma is characterized by a poor prognosis and resistance to conventional therapies. Infiltration of tumor-associated macrophages (TAM) is prominent in mesothelioma and is linked to immune suppression, angiogenesis, and tumor aggressiveness. Therefore, TAM depletion could potentially reactivate antitumor immunity. We show that M-CSFR inhibition using the CSF-1R kinase inhibitor PLX3397 (pexidartinib) effectively reduced numbers of TAMs, circulating nonclassical monocytes, as well as amount of neoangiogenesis and ascites in mesothelioma mouse models, but did not improve survival. When combined with dendritic cell vaccination, survival was synergistically enhanced with a concomitant decrease in TAMs and an increase in CD8+ T-cell numbers and functionality. Total as well as tumor antigen-specific CD8+ T cells in tumor tissue of mice treated with combination therapy showed reduced surface expression of the programmed cell death protein-1 (PD-1), a phenomenon associated with T-cell exhaustion. Finally, mice treated with combination therapy were protected from tumor rechallenge and displayed superior T-cell memory responses. We report that decreasing local TAM-mediated immune suppression without immune activation does not improve survival. However, combination of TAM-mediated immune suppression with dendritic cell immunotherapy generates robust and durable antitumor immunity. These findings provide insights into the interaction between immunotherapy-induced antitumor T cells and TAMs and offer a therapeutic strategy for mesothelioma treatment. Cancer Immunol Res; 5(7); 535-46. ©2017 AACR.

Ovarian cancer ascites induces a T cell suppressive phenotype in mature neutrophils: a potential barrier to anti-tumor immunity

Abstract Ovarian cancer (OC) is often diagnosed at advanced stages and presents with ascites. Necrosis is a hallmark of advanced cancer and releases DAMPs that activate innate immune responses (e.g., neutrophils). While neutrophils are critical in host defense, their role in the context of tumor is less understood. CTLs correlate with better outcomes in advanced OC, and immunotherapy for cancer is based on augmentation of CTL responses. Our prior studies showed that granulocytes from ascites of patients with newly diagnosed OC suppressed donor T cell proliferation ex vivo. The current work probes mechanisms for this suppression. In ascites from patients with newly diagnosed OC, ~90% of cells were inflammatory (CD45+). Neutrophils comprised ~15% of CD45+ cells and the neutrophil:CD8+ T cell ratio was 1.5:1. In ex vivo studies, cell-free ascites (ASC; 500g) attracted normal donor neutrophils (NDN). While neither ASC nor NDN alone impaired CD3/CD28-stimulated normal donor T cell proliferation, ASC (10/15 patients) when combined with NDN (1:1) completely suppressed T cell proliferation. Suppression by ASC + NDN required cell contact and was lost if NDN were added after overnight CD3/CD28 stimulation of T cells. Finally, heat-inactivating ASC (56C, 1h) abrogated the ability of NDN to suppress T cell proliferation. Here we provide evidence that ascites recruits neutrophils to the tumor microenvironment and induces a suppressive phenotype of neutrophils that requires cell contact and heat-labile cell-free constituents. Ongoing studies aim to identify the putative protein(s) in ascites and the signaling effects on neutrophils. Our long-term goal is to develop novel approaches to overcome this suppression and improve durable anti-tumor immunity.

Local Activation of p53 in the Tumor Microenvironment Overcomes Immune Suppression and Enhances Antitumor Immunity.

Mutations in tumor suppressor p53 remain a vital mechanism of tumor escape from apoptosis and senescence. Emerging evidence suggests that p53 dysfunction also fuels inflammation and supports tumor immune evasion, thereby serving as an immunological driver of tumorigenesis. Therefore, targeting p53 in the tumor microenvironment (TME) also represents an immunologically desirable strategy for reversing immunosuppression and enhancing antitumor immunity. Using a pharmacological p53 activator nutlin-3a, we show that local p53 activation in TME comprising overt tumor-infiltrating leukocytes (TILeus) induces systemic antitumor immunity and tumor regression, but not in TME with scarce TILeus, such as B16 melanoma. Maneuvers that recruit leukocytes to TME, such as TLR3 ligand in B16 tumors, greatly enhanced nutlin-induced antitumor immunity and tumor control. Mechanistically, nutlin-3a-induced antitumor immunity was contingent on two nonredundant but immunologically synergistic p53-dependent processes: reversal of immunosuppression in the TME and induction of tumor immunogenic cell death, leading to activation and expansion of polyfunctional CD8 CTLs and tumor regression. Our study demonstrates that unlike conventional tumoricidal therapies, which rely on effective p53 targeting in each tumor cell and often associate with systemic toxicity, this immune-based strategy requires only limited local p53 activation to alter the immune landscape of TME and subsequently amplify immune response to systemic antitumor immunity. Hence, targeting the p53 pathway in TME can be exploited to reverse immunosuppression and augment therapeutic benefits beyond tumoricidal effects to harness tumor-specific, durable, and systemic antitumor immunity with minimal toxicity. Cancer Res; 77(9); 2292-305. ©2017 AACR.

LIGHT Elevation Enhances Immune Eradication of Colon Cancer Metastases.

The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

Background.Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511–expressing gliomas in a syngeneic mouse model.Methods.Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC.Results.Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma–expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell–dependent protection from future tumor challenge.Conclusions.Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

Abstract B50: Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors

Abstract Development of effective and durable antitumor immunity requires the activation, expansion and maintenance of function of tumor antigen-specific effector T cells. The mechanism of action of recently FDA approved monoclonal antibody (mAb) therapies which target immune checkpoints such as PD-1 and CTLA-4 and uncouple inhibitory pathways from the activation of antigen-specific T cells underlines the critical requirement for antigen-specific priming to elicit potent and long-lasting anti-tumor immunity. However, immune checkpoint blockade results in significant clinical benefit largely in malignancies with high mutational burden, where the tumor itself can initiate T cell priming, but is less effective when used as a single agent among cancers with lower mutational burden, associated typically with a low level of lymphocyte infiltration into the tumor microenvironment (TME). We are evaluating clinical immunotherapy regimens combining immune checkpoint blockade with recombinant live-attenuated double-deleted strains of the intracellular bacterium Listeria monocytogenes (LADD), based on a hypothesis that effective immunotherapy will result from agents that in combination re-polarize the TME to facilitate immune effector cell function, prime functional tumor-specific T cells in the appropriate context, and block inhibitory signaling pathways. Here we show that tumor antigen-expressing LADD therapy enhanced CD8+ T cell effector function, resulting in significant tumor eradication in several preclinical mouse models. LADD treatment in addition induced favorable changes in the TME, as shown by enhanced CD8+ T effector function, recruitment of antigen presenting cells and reduction of regulatory T cells. Treatment regimens combining LADD-based immunotherapy with PD-1 immune checkpoint blockade significantly enhanced antitumor efficacy in CT26, 4T1 and MC38 tumor models. Together these data support the rationale for integrating LADD-based immunotherapy into clinical regimens with immune checkpoint blockade on the basis that TME modification and priming of tumor Ag-specific T cells significantly enhances the activity of mAb therapies blocking T cell inhibitory pathways. Citation Format: Weiwen Deng, Weiqun Liu, Thomas Hudson, Chris S. Rae, Ed Lemmens, Anthony Desbien, Bill Hanson, Pete Lauer, Thomas W. Dubensky, Jr., Meredith Leong. Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B50.

Intratumoral G100 Rescues Radiation-Induced T Cell Depletion and Has Synergistic Anti-Tumor Effect with Local Irradiation in A20 Lymphoma

Background: Radiation therapy is a standard established treatment modality for many malignancies, including lymphoma. There is growing evidence that the combination of radiation and immunotherapies can have synergistic immune and clinical effects; however, the mechanisms underlying these responses are poorly understood.Hypothesis: We hypothesized that the potential beneficial effect of adding immunotherapy to radiation might be due to alteration of the tumor microenvironment (TME) to an inflammatory phenotype and the facilitation of T-cell infiltration.Methods: Mice received two bilateral subcutaneous implant of 5 million A20 lymphoma cells each. When tumors are established (average tumor size = 45mm2, around Day 7~11), mice received intratumoral injection of G100, a synthetic TLR4 agonist, GLA-SE, tumor irradiation, or the combination of G100 and irradiation. X-ray radiation was delivered by using a Small Animal Radiation Research Platform (SARRP) with CT guided imaging that can accurately target lesions. Mice received a single dose of 10 Gy at one of the tumor implants. Intratumoral injection of G100 (10ug) was administered 3 times a week for up to 3 weeks, starting on the day post radiation.Results: The combination of G100 and radiation had synergistic antitumor effects and demonstrated an abscopal effect with tumor growth delay in the non-treated contralateral tumor implant. Long-term tumor immunity was also demonstrated as the surviving mice rejected secondary tumor challenge. Study of the TME at gene expression and cellular levels showed that G100 induces a pro-inflammatory cytokine and chemokine milieu that induced T cell trafficking into the tumors. G100-treated tumors showed increased infiltration of T cells, including both CD4 and CD8 T cells. In contrast, tumors from animals that received only irradiation but no G100 had significantly decreased levels of T lymphocytes as compared to untreated tumors (9.5±2.0% T cells in irradiated tumors vs. 32.0±5.0% in untreated tumors, p=0.0076), due to the cytotoxic effect of radiation on lymphocytes. CD4 tumor infiltrating lymphocytes (TILs) were depleted by 72% (6.8±2.2% vs. 24.4±4.6%, p=0.02) and CD8 TIL were depleted by 77% (1.5±0.3% vs. 6.6±0.7%, p=0.0004) in irradiated mice as compared to untreated mice. In the irradiation and G100 combination treatment group, the tumors had levels of T cells that were close to non-irradiated animals.Conclusion: The anti-tumor effect of irradiation is synergistically enhanced by concomitant intratumoral treatment by G100, which also results in abscopal responses and durable anti-tumor immunity. The modulation of the TME by G100 after irradiation also rescues the T-cell depletion caused by irradiation allowing for an influx of T cells. These preclinical results support the rationale for an on-going Phase 1/2 clinical trial in low-grade follicular non-Hodgkin's lymphoma (NHL) patients, who are randomized to receive G100 following local radiation with or without pembrolizumab (NCT02501473). DisclosuresLu:Immune Design: Employment. Hewitt:Immune Design: Employment. Hsu:Immune Design: Employment. ter Meulen:Immune Design: Employment.

Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer

ABSTRACTThere is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4+ and CD8+ effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer.

Compensatory upregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agent checkpoint blockade in metastatic ovarian cancer

ABSTRACTTumor-associated or -infiltrating lymphocytes (TALs or TILs) co-express multiple immune inhibitory receptors that contribute to immune suppression in the ovarian tumor microenvironment (TME). Dual blockade of PD-1 along with LAG-3 or CTLA-4 has been shown to synergistically enhance T-cell effector function, resulting in a delay in murine ovarian tumor growth. However, the mechanisms underlying this synergy and the relative contribution of other inhibitory receptors to immune suppression in the ovarian TME are unknown. Here, we report that multiple immune checkpoints are expressed in TALs and TILs isolated from ovarian tumor-bearing mice. Importantly, blockade of PD-1, LAG-3, or CTLA-4 alone using genetic ablation or blocking antibodies conferred a compensatory upregulation of the other checkpoint pathways, potentiating their capacity for local T-cell suppression that, in turn, could be overcome through combinatorial blockade strategies. Whereas single-agent blockade led to tumor outgrowth in all animals, dual antibody blockade against PD-1/CTLA-4 or triple blockade against PD-1/LAG-3/CTLA-4 resulted in tumor-free survival in 20% of treated mice. In contrast, dual blockade of LAG-3 and CTLA-4 pathways using PD-1 knockout mice led to tumor-free survival in 40% of treated mice, suggesting a hierarchical ordering of checkpoint function. Durable antitumor immunity was most strongly associated with increased numbers of CD8+ T cells, the frequency of cytokine-producing effector T cells, reduced frequency of Tregs and arginine-expressing monocytic myeloid-derived suppressor cells in the peritoneal TME. These data provide a basis for combinatorial checkpoint blockade in clinical intervention for ovarian cancer.

Chemo-immunotherapy mediates durable cure of orthotopic KrasG12D/p53−/− pancreatic ductal adenocarcinoma

ABSTRACTPancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, exhibiting a five-year overall survival (OS) of only 7% despite aggressive standard of care. Recent advances in immunotherapy suggest potential application of immune-based treatment approaches to PDAC. To explore this concept further, we treated orthotopically established K-rasG12D/p53−/− PDAC tumors with gemcitabine and a cell-based vaccine previously shown to generate durable cell-mediated (TH1) immunity. Tumor progression was monitored by IVIS. The results indicated that the combination of chemotherapy and dendritic cell (DC) vaccination was effective in eliminating tumor, preventing metastasis and recurrence, and significantly enhancing OS. No animal that received the combination therapy relapsed, while mice that received gemcitabine-only or vaccine-only regimens relapsed and progressed. Analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of CD8+IFNγ+CCR7+NK1.1+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells after the cessation of treatment. Retro-orbital tumor re-challenge of surviving animals at six-months post-treatment demonstrated durable antitumor immunity only among mice that had received the combination therapy. CD8+ splenocytes derived from surviving mice that had received the combination therapy were sorted into NK1.1pos and NK1.1neg populations and adoptively transferred into naive recipients. Transfer of only 1,500 CD8+NK1.1pos T-cells was sufficient to mediate tumor rejection whereas transfer of 1,500 CD8+NK1.1neg T-cells imparted only minimal effects. The data suggest that addition of a TH1 DC vaccine regimen as an adjuvant to existing therapies can mediate eradication of tumors and offer durable protection against PDAC.