Durable Immune Responses Research Articles

Metabolic Stress in the Immune Function of T Cells, Macrophages and Dendritic Cells.

Innate and adaptive immune cells from myeloid and lymphoid lineages resolve host infection or cell stress by mounting an appropriate and durable immune response. Upon sensing of cellular insults, immune cells become activated and undergo rapid and efficient functional changes to unleash biosynthesis of macromolecules, proliferation, survival, and trafficking; unprecedented events among other mammalian cells within the host. These changes must become operational within restricted timing to rapidly control the insult and to avoid tissue damage and pathogen spread. Such changes occur at high energy cost. Recent advances have established that plasticity of immune functions occurs in distinct metabolic stress features. Evidence has accumulated to indicate that specific metabolic signatures dictate appropriate immune functions in both innate and adaptive immunity. Importantly, recent studies have shed light on whether successfully manipulating particular metabolic targets is sufficient to modulate immune function and polarization, thereby offering strong therapeutic potential for various common immune-mediated diseases, including inflammation and autoimmune-associated diseases and cancer. In this review, we detail how cellular metabolism controls immune function and phenotype within T cells and macrophages particularly, and the distinct molecular metabolic programming and targets instrumental to engage this regulation.

A Multiagent Alphavirus DNA Vaccine Delivered by Intramuscular Electroporation Elicits Robust and Durable Virus-Specific Immune Responses in Mice and Rabbits and Completely Protects Mice against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenges.

There remains a need for vaccines that can safely and effectively protect against the biological threat agents Venezuelan (VEEV), western (WEEV), and eastern (EEEV) equine encephalitis virus. Previously, we demonstrated that a VEEV DNA vaccine that was optimized for increased antigen expression and delivered by intramuscular (IM) electroporation (EP) elicited robust and durable virus-specific antibody responses in multiple animal species and provided complete protection against VEEV aerosol challenge in mice and nonhuman primates. Here, we performed a comparative evaluation of the immunogenicity and protective efficacy of individual optimized VEEV, WEEV, and EEEV DNA vaccines with that of a 1 : 1 : 1 mixture of these vaccines, which we have termed the 3-EEV DNA vaccine, when delivered by IM EP. The individual DNA vaccines and the 3-EEV DNA vaccine elicited robust and durable virus-specific antibody responses in mice and rabbits and completely protected mice from homologous VEEV, WEEV, and EEEV aerosol challenges. Taken together, the results from these studies demonstrate that the individual VEEV, WEEV, and EEEV DNA vaccines and the 3-EEV DNA vaccine delivered by IM EP provide an effective means of eliciting protection against lethal encephalitic alphavirus infections in a murine model and represent viable next-generation vaccine candidates that warrant further development.

Optimal use of meningococcal serogroup B vaccines: moving beyond outbreak control.

Neisseria meningitidis is a major cause of meningitis and septicemia globally. Vaccines directed against N. meningitidis serogroup B (MenB) have been used to control sporadic and sustained disease in industrialized and non-industrialized countries. Early outer membrane vesicle (OMV) vaccines effectively reduced MenB disease in countries such as Norway, New Zealand, and France; however, these vaccines were highly specific for their targeted outbreak strain, did not elicit a durable immune response, and were ineffective for widespread use due to the diversity of MenB-disease-causing isolates. Recently developed recombinant protein-based MenB vaccines that target conserved surface proteins have the potential to induce a broader immune response against the diversity of disease-causing strains. Given the deleterious consequences and sporadic nature of MenB disease, the use of optimal vaccination strategies is crucial for prevention. Reactive vaccination strategies used in the past have significant limitations, including delayed implementation, substantial use of resources, and time constraints. The broad coverage potential of recombinant protein-based MenB vaccines suggests that routine use could result in a reduced burden of disease. Despite this, routine use of MenB vaccines is currently limited in practice.

Dendritic cell vaccine (DCVAC) with chemotherapy (ct) in patients (pts) with epithelial ovarian carcinoma (EOC) after primary debulking surgery (PDS): Interim analysis of a phase 2, open-label, randomized, multicenter trial.

5509Background: Most pts with EOC relapse after PDS and ct. Autologous DCVAC can present tumor antigens to elicit a durable immune response. We hypothesized that adding DCVAC to ct could improve ou...

Identification and characterization of novel mediators of tumor-induced T-cell dysfunction

Abstract Immunotherapies targeting T-cell functionality, such as anti-PD-1, have been shown to have efficacy in numerous cancers, including non-small cell lung cancer (NSCLC). However, treatment with anti-PD-1 induces responses in only a subset of patients. This raises the possibility that other effectors of T-cell dysfunction remain to be elucidated. To evaluate possible novel mediators of T-cell dysfunction, we isolated CD8+ T-cells from orthotopic KrasG12D/p53−/− murine lung adenocarcinomas (HKP-1) that exhibited either stable or progressive disease as a function of tumor growth, both in the presence and absence of anti-PD-1 therapy. RNAseq analysis revealed increased expression of numerous genes including Tim3, PD-1, Lag3, 2B4, and CISH, and decreased expression of GzmB and Eomes in CD8+ T-cells isolated from progressive tumors indicative of a dysfunctional state, as well as multiple novel genes not previously associated with T-cell dysfunction. To explore the functional role of the novel candidate genes, we have developed in vitro and in vivo assays evaluating cytotoxicity, cytokine production, proliferation, and tumor growth, leveraging Ova expression in our HKP-1 cell line (KP1-Ova). We have begun to target this unique set of candidate genes associated with dysfunction in tumor-specific T-cells through RNAi/shRNA and adoptive transfer approaches to facilitate therapeutic reprogramming and enhanced T-cell-mediated immunity in lung cancer. We expect to provide novel strategies for designing rational immunotherapies targeting dysfunctional tumor-specific CD8+ T-cells to generate durable anti-tumor immune responses for the treatment of lung adenocarcinoma.

Clay Nanoparticles Elicit Long-Term Immune Responses by Forming Biodegradable Depots for Sustained Antigen Stimulation.

Nanomaterials have been widely tested as new generation vaccine adjuvants, but few evoke efficient immunoreactions. Clay nanoparticles, for example, layered double hydroxide (LDH) and hectorite (HEC) nanoparticles, have shown their potent adjuvanticity in generating effective and durable immune responses. However, the mechanism by which clay nanoadjuvants stimulate the immune system is not well understood. Here, it is demonstrated that LDH and HEC-antigen complexes form loose agglomerates in culture medium/serum. They also form nodules with loose structures in tissue after subcutaneous injection, where they act as a depot for up to 35 d. More importantly, clay nanoparticles actively and continuously recruit immune cells into the depot for up to one month, and stimulate stronger immune responses than FDA-approved adjuvants, Alum and QuilA. Sustained antigen release is also observed in clay nanoparticle depots, with 50-60% antigen released after 35 d. In contrast, Alum-antigen complexes show minimal antigen release from the depot. Importantly, LDH and HEC are more effective than QuilA and Alum in promoting memory T-cell proliferation. These findings suggest that both clay nanoadjuvants can serve as active vaccine platforms for sustained and potent immune responses.

Effective cancer immunotherapy in mice by polyIC-imiquimod complexes and engineered magnetic nanoparticles

Encouraging results are emerging from systems that exploit Toll like receptor (TLR) signaling, nanotechnology, checkpoint inhibition and molecular imaging for cancer immunotherapy. A major remaining challenge is developing effective, durable and tumour-specific immune responses without systemic toxicity. Here, we report a simple and versatile system based on synergistic activation of immune responses and direct cancer cell killing by combined TLR ligation using polyIC as TLR3 and imiquimod (R837) as TLR7 agonist, in combination with the model antigen ovalbumin (OVA) and phospholipid micelles loaded with zinc-doped iron oxide magnetic nanoparticles (MNPs). The combination of TLR agonists triggered a strong innate immune response in the lymph nodes (LNs) without systemic release of pro-inflammatory cytokines. The vaccines showed excellent efficacy against aggressive B16-F10 melanoma cells expressing OVA, which was improved with immune checkpoint abrogation of the immunosuppressive programmed death-ligand 1 (PD-L1) at the level of the cancer cells. By magnetic resonance (MR) and nuclear imaging we could track the vaccine migration from the site of injection to LNs and tumour. Overall, we show this synergistic TLR agonists and their combination with MNPs and immune checkpoint blockade to have considerable potential for preclinical and clinical development of vaccines for cancer immunotherapy.

Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against Zika Virus Infection and Testis Damage.

The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitoes, as seen for classical arboviruses. We along with others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available; therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low preexisting immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week postvaccination, and the elicited antiserum could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mouse sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control.IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barré syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid preexisting anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M/E glycoproteins was used for ZIKV vaccine development. Impressively, AdC7-M/E exhibited exceptional performance as a ZIKV vaccine, as follows: (i) protective efficacy by a single vaccination, (ii) rapid development of a robust humoral response, (iii) durable immune responses, (iv) robust T cell responses, and (v) sterilizing immunity achieved by a single vaccination. These advantages of AdC7-M/E strongly support its potential application as a promising ZIKV vaccine in the clinic.

Nanocage-Therapeutics Prevailing Phagocytosis and Immunogenic Cell Death Awakens Immunity against Cancer.

A growing appreciation of the relationship between the immune system and the tumorigenesis has led to the development of strategies aimed at "re-editing" the immune system to kill tumors. Here, a novel tactic is reported for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, but also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induce the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. Potent inhibition of tumor growth and complete eradication of tumors is observed through systemic tumor-specific T cell responses in tumor draining lymph nodes and the spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics "awakens" the host's immune system and provokes a durable systemic immune response against cancer.

Abstract A201: Discovery of TSR-033, a novel, potent anti-human LAG-3 therapeutic antibody

Abstract Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on immune cells. Similar to other immune inhibitory receptors such as programmed death-1 (PD-1), LAG-3 ligation suppresses T-cell activation and cytokine production following antigen stimulation. Despite favorable response rates observed with PD-1 or PD-1 ligand blockade in the treatment of human cancers, there remain many patients who either derive little clinical benefit from such therapies (primary resistance) or suffer a relapse (acquired or adaptive immune resistance). LAG-3 has been implicated in tumor extrinsic resistance mechanisms, due in part to its upregulation following PD-1 blockade, suggesting that targeting this pathway in addition to PD-1 may engender stronger and more durable antitumor immune responses. TSR-033 is a potent and selective humanized monoclonal antibody of the IgG4/kappa isotype, generated through the use of a proprietary in vitro somatic hypermutation antibody discovery technology platform. TSR-033 binds to human LAG-3 and blocks the interaction between LAG-3 and its ligand, major histocompatibility complex class II, expressed on Daudi cells. When evaluated for C1q and CD16a binding, TSR-033 displays properties typical of a human IgG4 antibody, suggesting that it is unlikely to mediate appreciable complement-dependent cytotoxicity or antibody-dependent cell cytotoxicity. Functionally, TSR-033 enhanced T-cell activation in vitro in both SEB-driven and mixed lymphocyte reaction assays. The combination of TSR-033 with a ligand blocking anti-PD-1 antibody further augmented T-cell activation in both systems, as evidenced by increased cytokine production, relative to each individual reagent. Analogous studies using surrogate anti-mouse antibodies also demonstrated that combined blockade of PD-1 and LAG-3 significantly boosted IFNγ production in an in vitro model of murine CD4 T cell exhaustion. Furthermore, anti-LAG-3 combined with anti-PD-1 improved therapeutic efficacy in mouse syngeneic tumor models, eliciting durable immunologic memory upon tumor rechallenge and altering intratumoral and splenic T-cell populations, consistent with heightened immune activation. Taken together, the pharmacologic activity of TSR-033 demonstrates that it is a potent anti-LAG-3 therapeutic antibody and supports its clinical investigation in cancer patients. Citation Format: Srimoyee Ghosh, Haley Laken, Jon Travers, Sujatha Kamar, H. Toni Jun, Marilyn R. Kehry, David Jenkins. Discovery of TSR-033, a novel, potent anti-human LAG-3 therapeutic antibody [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 A201.

Targeting autophagy inhibits melanoma growth by enhancing NK cells infiltration in a CCL5-dependent manner.

While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

The renaissance of anti-neoplastic immunity from tumor cell demise.

Cancer therapies can temporarily reduce tumor burdens by inducing malignant cell death. However, cancer cure is still far from realization because tumors often gain resistance to current treatment and eventually relapse. Accumulating evidence suggests that successful cancer interventions require anti-tumor immunity. Therapy-induced cell stress responses ultimately result in one or more cell death modalities, including apoptosis, autophagy, necroptosis, and pyroptosis. These irreversible dying processes are accompanied by active or passive release of cell death-associated molecular patterns (CDAMPs), which can be sensed by corresponding pattern recognition receptors (PRR) on tumor-infiltrating immune cells. This crosstalk with the immune system can reawaken immune surveillance in the tumor microenvironment (TME). This review focuses on immune-modulatory properties of anti-cancer regimens and CDAMP-mediated communications between cell stress responses and the immune contexture of TME. In addition, we describe how immunogenic cell death can elicit strong and durable anti-tumor immune responses.

Galinpepimut-S, a WT1-Targeting Immuno-Oncology Treatment, Induces Specific, Robust and Durable Immune Responses (IRs) in Patients (Pts) with High-Risk (HR) Multiple Myeloma (MM)

Galinpepimut-S, a WT1-Targeting Immuno-Oncology Treatment, Induces Specific, Robust and Durable Immune Responses (IRs) in Patients (Pts) with High-Risk (HR) Multiple Myeloma (MM)

Protocol of a randomised controlled trial characterising the immune responses induced by varicella-zoster virus (VZV) vaccination in healthy Kenyan women: setting the stage for a potential VZV-based HIV vaccine

IntroductionA protective HIV vaccine would be expected to induce durable effector immune responses at the mucosa, restricting HIV infection at its portal of entry. We hypothesise that use of varicella-zoster...

Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma.

In the last few years, there has been a twist in cancer treatment toward immunotherapy thanks to the impressive results seen in advanced patients from several tumor pathologies. Cutaneous melanoma is a highly mutated and immunogenic tumor that has been a test field for the development of immunotherapy. However, there is still a way on the road to achieving complete and long-lasting responses in most patients. It is desirable that immunotherapeutic strategies induce diverse immune reactivity specific to tumor antigens, including the so-called neoantigens, as well as the blockade of immunosuppressive mechanisms. In this review, we will go through the role of promising monoclonal antibodies in cancer immunotherapy with immunomodulatory function, especially blocking of the inhibitory immune checkpoints CTLA-4 and PD-1, in combination with different immunotherapeutic strategies such as vaccines. We will discuss the rational basis for these combinatorial approaches as well as different schemes currently under study for cutaneous melanoma in the clinical trials arena. In this way, the combination of “push and release” immunomodulatory therapies can contribute to achieving a more robust and durable antitumor immune response in patients.

The immune contexture in cancer prognosis and treatment.

Immunotherapy is currently the most rapidly advancing area of clinical oncology, and provides the unprecedented opportunity to effectively treat, and even cure, several previously untreatable malignancies. A growing awareness exists of the fact that the success of chemotherapy and radiotherapy, in which the patient's disease can be stabilized well beyond discontinuation of treatment (and occasionally is cured), also relies on the induction of a durable anticancer immune response. Indeed, the local immune infiltrate undergoes dynamic changes that accompany a shift from a pre-existing immune response to a therapy-induced immune response. As a result, the immune contexture, which is determined by the density, composition, functional state and organization of the leukocyte infiltrate of the tumour, can yield information that is relevant to prognosis, prediction of a treatment response and various other pharmacodynamic parameters. Several complementary technologies can be used to explore the immune contexture of tumours, and to derive biomarkers that could enable the adaptation of individual treatment approaches for each patient, as well as monitoring a response to anticancer therapies.

HIV Env conserved element DNA vaccine alters immunodominance in macaques

ABSTRACTSequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of ‘conserved’ and ‘variable’ regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0–5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines.

Abstract 643: Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth

Abstract We recently reported that the aberrant expression of Major Histocompatibility Class II (MHCII) molecules on human triple negative breast cancer (TNBC) cells correlates with prolonged progression-free survival and increased tumor infiltrating lymphocytes. We hypothesized that the expression of MHCII enhances the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, resulting in more effective tumor control. To test our hypothesis, we created both MHCII-expressing and MHCII-negative tumor cells by transfecting murine breast cancer (TS/A) cells with the human class II transcriptional activator (hCIITA) or empty vector, respectively. Transfected cells were then injected into BALB/c mice and the resulting immune response analyzed by flow cytometry at four time points. We found that hCIITA-expressing tumors grew slower than control tumors in immunocompetent recipients, but that this difference was nullified in immunocompromised and markedly reduced in CD4+ T cell depleted mice. CD4+ T cells isolated from hCIITA-transfected tumors produced more IFNγ, IL-17A, and surprisingly granzyme B for longer times than their counterparts in control tumors. Similarly, CD8+ T cells isolated from hCIITA-transfected tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to enhanced effector functions, TCR repertoire analysis demonstrated that both the breadth and magnitude of expansion of responding T cell clones were increased in hCIITA-transfected tumors. Interestingly, TS/A-hCIITA tumors harbored more regulatory T cells (Tregs) with a more suppressive phenotype than Tregs from control tumors. Finally, we show that the histone deacetylase inhibitor (HDACi) Entinostat is capable of robust and dose-dependent induction of MHCII on tumor cells in vivo, an effect that correlates with dramatic reduction in tumor size. These results suggest that the clinical benefit associated with MHCII expression on TNBC cells is mediated by a delay in T cell exhaustion and increased intratumoral CD4+ T cell activation, which enhances the cytotoxic capacity of CD8+ T cells. Entinostat, and potentially other epigenetic modifying agents, may enable induction of MHCII expression on TNBC cells clinically and allow more patients to benefit from an augmented T cell response. These effects may be magnified by combinatorial therapy with checkpoint inhibitors to promote durable anti-tumor immune responses. Citation Format: Tyler R. McCaw, Mei Li, Selene Meza-Perez, Donald J. Buchsbaum, Dmytro Starenki, Sara Cooper, Andres Forero, Troy D. Randall. Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth [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 643. doi:10.1158/1538-7445.AM2017-643

Abstract 5659: Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer

Abstract Pancreatic cancer remains a serious unmet medical need. The majority of pancreatic tumors are composed of stromal cells that make tumors resistant to conventional therapies. We are developing an approach in which a TLR9 agonist, IMO-2125, is administered intratumorally, which induces local Th1 responses and modulates the tumor microenvironment. This approach has shown promising antitumor activity in preclinical models of multiple tumor types in combination with checkpoint inhibitors. We are employing a similar approach to the treatment of pancreatic cancer. We evaluated the local therapeutic effect of IMO-2125 in a murine syngeneic pancreatic ductal adenocarcinoma (Panc02) cancer models. In the first study, C57BL/6 mice were intraperitoneally (i.p.) implanted with Panc02 cells to generate peritoneal metastatic cancer. Tumor-bearing mice (n=10 per group) were then treated with 2.5 mg/kg IMO-2125 either by i.p. or subcutaneous (s.c.) injections for six times in two weeks. In the second study, to establish dual solid tumors in C57BL/6 mice, Panc02 cells were subcutaneously implanted in both the right and left flanks of the animals. When tumors reached to 50-150 mm3, we started the intratumoral (i.t.) administration of IMO-2125 in right tumors at doses of either 0.25 mg/kg, 1 mg/kg or 2.5 mg/kg, or PBS (n=10 per group), 3x/week for three weeks. In the peritoneal metastatic pancreatic tumor model, 80% of IMO-2125 intraperitoneally treated mice survived for more than 88 days, while 90% of the control group died within 29 days. Compared to i.p. treatment, the s.c. treated group survived for only 36 days. Tumor-free mice from the IMO-2125 i.p treatment group (n=7) rejected the same tumor rechallenge, suggesting establishment of durable tumor-specific immune response. In the dual solid pancreatic tumor model, intratumoral IMO-2125 treatment led to a dose dependent antitumor activity in both injected and distant tumors. Tumors treated with 0.25, 1 and 2.5 mg/kg doses of IMO-2125 showed reductions in tumor volume by 66% (p = 0.004), 74% (p = 0.002) and 95% (p = 0.003), respectively. Moreover, the distant tumors exhibited a reduction of 0% (p = 0.65), 64% (p = 0.006) and 69% (p = 0.001), respectively. Antitumor activity was associated with increased T-cell infiltration and upregulation of the gene expression of various checkpoints in both treated and distant tumors. In an independent study, treatment with a control IMO showed minimal antitumor activity. All treatments were well tolerated. In summary, local treatment with IMO-2125 exerts antitumor activity in preclinical pancreatic cancer models, associated with an increase in T-cell infiltration in the tumor microenvironment, providing a novel approach to the treatment of pancreatic cancer. The clinical trial of intratumoral IMO-2125 is in progress in patients with melanoma. Citation Format: Daqing Wang, Evren Kocabas Argon, Fugang Zhu, Sudhir Agarwal. Local treatment with novel TLR9 agonist IMO-2125 demonstrates antitumor activity in preclinical models of pancreatic cancer [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 5659. doi:10.1158/1538-7445.AM2017-5659

Abstract 4691: Polarization of macrophages with STING agonists

Abstract The functional plasticity of macrophages and their ability to support tumour growth or promote potent antitumor immunity make them an attractive target cancer therapy. Macrophages have been implicated in the complex antitumor activity of murine STING agonist, DMXAA, including disruption of tumor vasculature, induction of cytokine production, and promotion of durable antitumor immune responses. Many of these activities are lost in STING-/- mice, and DMXAA does not bind to human STING, providing a pertinent explanation for why the anti-tumour effects of DMXAA observed in mice were not recapitulated human clinical trials. Using PMA-differentiated THP-1 macrophages, we sought to identify human active analogues of DMXAA in vitro, comparing their capacity to induce cytokines indicative of a classically activated antitumour phenotype; the non-canonical STING agonist 2'3'-cGAMP was also included in our investigation. IP-10 (CXCL10) production, measured by ELISA, was used as an initial marker for activity, followed by multiplex cytokine analysis for broader characterisation compared to M(IFN-γ/LPS), M(IL-4/IL-13), and ‘resting’ M(media alone) macrophage phenotypes. Parallel experiments were also conducted with RAW264.7 murine macrophages to compare the DMXAA-induced cytokine profile directly with that of 2'3'-cGAMP. 2'3'-cGAMP induced 22-fold and 18-fold increases in IP-10 production in resting and M(IL-4/IL-13) THP-1 macrophages, respectively. By comparison, THP-1 macrophages were much less responsive to DMXAA and analogues tested to date. Furthermore, 2'3'-cGAMP induced a spectrum of cytokines similar to that observed in M(IFN-γ/LPS) macrophages, and markedly different to that of M(IL-4/IL-13) THP-1 macrophages. DMXAA, and analogues previously shown to have cytokine-inducing activity in human leukocytes did not produce cytokine spectra indicative of a classic M(IFN-γ/LPS) phenotype. Unlike human cells, however, cytokine spectra produced by RAW264.7 murine macrophages were comparable between DMXAA- and 2'3'-cGAMP-treated cells. These findings support previous data that suggest that STING agonists can promote macrophage polarisation towards an antitumour phenotype. While DMXAA is active in murine macrophages, only 2'3'-cGAMP can induce in human THP-1 cells, an M(IFN-γ/LPS)-like spectrum of cytokines often attributed to an antitumour macrophage phenotype. Citation Format: Kimiora L. Henare, Sofian M. Tijono, Lai-Ming Ching. Polarization of macrophages with STING agonists [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 4691. doi:10.1158/1538-7445.AM2017-4691