Enhanced Antigen Presentation Research Articles

Abstract 4549: Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms

Abstract Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [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 4549.

Abstract 4043: A novel role for Hedgehog signaling in macrophage-mediated immune evasion

Abstract In the tumor microenvironment, breast cancer cells participate in crosstalk with the surrounding stroma. This tumor-stromal interaction forms a balance that dictates tumor suppressing or promoting response mechanisms. Macrophages in the tumor microenvironment are plastic and can mediate several functions depending on their activation states. Tumor-associated macrophages co-exist as two major phenotypes: anti-tumorigenic and immune-eliciting classically activated M1 as well as tumor-promoting and immune-suppressive alternatively activated M2 macrophages. Alternatively activated macrophages are associated with more aggressive stages and poor clinical outcomes in breast cancer patients as they suppress the tumoricidal properties of the immune system, thus facilitating tumor cell proliferation and dissemination. Hedgehog (Hh) signaling is a critical developmental pathway; its activation modulates vital cellular processes such as cell proliferation, stem-cell differentiation, and angiogenesis. While the Hh signaling pathway is normally tightly controlled, it can often be dysregulated which fosters tumorigenesis and tumor progression. Aberrant Hh signaling activation is particularly implicated in breast cancer progression and metastasis. In this study, we investigate the role of Hh signaling in polarizing breast cancer-associated macrophages toward the detrimental M2 subtype. We report that targeting different steps of the Hh signaling cascade attenuates the cytokine profile associated with M2 macrophages. Additionally, we demonstrate that Hh signaling enables the molecular mechanism responsible for alternative macrophage polarization. We furthermore identify significant shifts in immune cell populations infiltrating the primary tumor when we administer an FDA-approved Hh inhibitor to an in vivo mammary tumor model. This altered immune profile is dynamically characterized by a reduction in immune-suppressive cells concomitant with enhanced antigen presentation properties and cytotoxic immune cell infiltration, overall culminating in reduced metastasis. Thus, we describe a novel role for Hh signaling in enabling pro-tumorigenic immunity by alternatively polarizing breast cancer-associated macrophages. This study identifies a novel strategy, which has potential clinical implications to treat breast cancer through targeting aberrant tumor cell Hh activation and eliciting a robust anti-tumorigenic immune response. Citation Format: Ann Hanna, Dongquan Chen, Darshan S. Chandrashekar, Sooryanarayana Varambally, Rajeev S. Samant, Lalita A. Shevde. A novel role for Hedgehog signaling in macrophage-mediated immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4043.

Immune suppression and reversal of the suppressive tumor microenvironment.

Most tumors employ multiple strategies to attenuate T-cell-mediated immune responses. In particular, immune suppression surrounding the tumor is achieved by interfering with antigen-presenting cells and effector T cells. Controlling both the tumor and the tumor microenvironment (TME) is critical for cancer treatment. Checkpoint blockade therapy can overcome tumor-induced immune suppression, but more than half of the patients fail to respond to this treatment; therefore, more effective cancer immunotherapies are needed. Generation of an anti-tumor immune response is a multi-step process of immune activation against the tumor that requires effector T cells to recognize and exert toxic effects against tumor cells, for which two strategies are employed-inhibition of various types of immune suppressor cells, such as myeloid cells and regulatory T cells, and establishment of anti-tumor immune surveillance including, activation of natural killer cells and cytotoxic T cells. It was recently shown that anti-cancer drugs not only directly kill tumor cells, but also influence the immune response to cancer by promoting immunogenic cell death, enhancing antigen presentation or depleting immunosuppressive cells. Herein, we review the mechanisms by which tumors exert immune suppression as well as their regulation. We then discuss how the complex reciprocal interactions between immunosuppressive and immunostimulatory cells influence immune cell dynamics in the TME. Finally, we highlight the new therapies that can reverse immune suppression in the TME and promote anti-tumor immunity.

Anti-viral immune response in the lung and thymus: Molecular characterization and expression analysis of immunoproteasome subunits LMP2, LMP7 and MECL-1 in pigs

Both the lung and the thymus are vital target organ for pathogens including viruses. The immunoproteasome (i-proteasome) enhances antigen presentation for MHC class I molecules to activate CD8+T lymphocyte. These facilitate antiviral adaptive immune response. Our previous study found that, expression of i-proteasome subunits in porcine lung was altered during normal and inflammatory conditions. To date, the expression of i-proteasome subunits in porcine thymus to viruses has not been investigated. In the present study, LMP2, LMP7, and MECL-1 were cloned, identified and their sequences encoded predicted proteins of 216, 275, and 278 amino acids, respectively. Expression of LMP2, LMP7, and MECL-1, in the cytoplasm and nucleus, was markedly altered in the porcine reproductive and respiratory syndrome virus (PRRSV)-infected lung and thymus. And dendritic cells and epithelial cells readily expressed the i-proteasome subunit LMP2 in the thymus of PRRSV-infected pigs compared to that in mock-infected pigs. Additionally, the in vitro stimulation of a PAM cell line with PolyI:C for 12 and 24 h resulted in increased LMP2, LMP7, and MECL-1 expression. These results suggest a central role for these complexes in the activation of an antiviral immune response in pigs. A better understanding of the role of the i-proteasome in different cell types, tissues, and hosts could improve vaccine design and facilitate the development of effective treatment strategies for viral infections.

Novel strategy to enhance antigen presentation and CD8 T cell immunity for curative efficacy of intranasal therapeutic HPV peptide vaccination in preclinical vaginal HPV tumor model

Abstract Immune checkpoint modulation to enhance antitumor immunity while minimizing immune suppressive responses within tumors has proven to be a highly successful cancer treatment strategy, but, often with toxic side effects and financial constraints. Using preclinical models of human papillomavirus (HPV) cancers, we tested vaccination employing adjuvants with diverse modes of action to promote antigen presentation and T cell immunity. We employed the combination of α-galactosylceramide (α-GalCer), a potent inducer of NKT cells and CpG-containing oligo-deoxynucleotide (CpG-ODN) that function through the TLR9 pathway. Since most HPV tumors occur at mucosal tissues, we used the mucosal intranasal route for vaccination with E6 and E7 peptides representing tumor-specific antigens to treat vaginal HPV+ TC-1 tumors in mice. Previously, we observed that vaccination using α-GalCer adjuvant alone reduced tumor growth but, complete and durable tumor regression required supplementing the vaccine with anti-4-1BB checkpoint modulating antibody that has clinical safety concerns. We now observed that intranasal HPV peptide vaccine incorporating α-GalCer and CpG-ODN adjuvants, rendered tumor-free survival advantage. Correlates of tumor protection included doubling the number of CD8 T cells exhibiting enhanced cytotoxic potential within the tumor and 84% increase in the ratio of CD8 T cells to regulatory T cells relative to vaccination with either adjuvant alone. Intranasal vaccination delivering α-GalCer plus CpG-ODN allows for the activation of dendritic cells through both classical and alternative licensing pathways and permits the secretion of a diverse set of chemokines to attract enhanced populations of naïve CD8+ T cells.

Augmenting vaccine immunogenicity through the use of natural human anti-Rha antibodies and monoclonal Fc domains

Abstract In order to obtain an effective cellular and humoral immune response, a vaccine construct has to be captured, processed and presented by antigen presenting cells (APCs) through their major histocompatibility complex (MHC) molecules. One effective way to enhance antigen presentation is through an antibody-dependent antigen uptake mechanism by targeting FcγR receptors of APCs that can bind with the Fc portion of an immune complexed antibody. Anti-Rhamnose (Anti-Rha) antibody is among the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. Binding rhamnose of a rhamnose-containing vaccine with murine anti-Rha antibody has previously been proven effective for capturing and processing of antigen by APCs via stimulatory Fc receptors. We show that human anti-Rha antibodies enhance the uptake of rhamnose-containing antigen by murine APCs in vitro. In-vivo, passive transfer of human anti-Rha antibodies into mice improves the priming of T cells to Rha-antigen. We synthesized and tested an anti-cancer vaccine containing a lipoglycopeptide cancer antigen in a Rha-bearing liposome and demonstrated improved immunogenicity. Also, we showed that direct conjugation of the Fc domain of an antibody to a liposomal vaccine instead of rhamnose can be a good approach for efficient uptake and presentation, as we can bypass the need for anti-Rha antibodies. A liposomal vaccine bearing IgG3Fc has been found to be equally effective as the Rha-vaccine at priming CD4+ T cells. Priming of CD8+ T cells for IFN-γ release and tumor cytotoxicity was promising in comparison to anti-Rha antibody targeting.

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and invitro assays to investigate the impact of abemaciclib on Tcells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased Tcell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a Tcell inflamed phenotype, and enhanced cell cycle control. Invitro, treatment with abemaciclib resulted in increased activation of human Tcells andupregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate Tcell anti-tumor immunity.

IL4 (interleukin 4) induces autophagy in B cells leading to exacerbated asthma

ABSTRACTAllergic asthma is a common airway inflammatory disease in which B cells play important roles through IgE production and antigen presentation. SNP (single nucleotide polymorphism) analysis showed that Atg (autophagy-related) allele mutations are involved in asthma. It has been demonstrated that macroautophagy/autophagy is essential for B cell survival, plasma cell differentiation and immunological memory maintenance. However, whether B cell autophagy participates in asthma pathogenesis remains to be investigated. In this report, we found that autophagy was enhanced in pulmonary B cells from asthma-prone mice. Autophagy deficiency in B cells led to attenuated immunopathological symptoms in asthma-prone mice. Further investigation showed that IL4 (interleukin 4), a key effector Th2 cytokine in allergic asthma, was critical for autophagy induction in B cells both in vivo and in vitro, which further sustained B cell survival and enhanced antigen presentation by B cells. Moreover, IL4-induced autophagy depended on JAK signaling via an MTOR-independent, PtdIns3K-dependent pathway. Together, our data indicate that B cell autophagy aggravates experimental asthma through multiple mechanisms.

Phase 1b/2 study of binimetinib (BINI) in combination with nivolumab (NIVO) or NIVO plus ipilimumab (IPI) in patients (pts) with previously treated microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) with RAS mutation.

TPS870 Background: Approximately 96% of CRCs have an MSS phenotype, which results in more immunologically quiescent tumors for which immunotherapies are largely ineffective (Lee et al. 2015; Overman et al. 2016). Pts with MSS CRC and activating RAS mutation (35%–45% of CRCs) have treatment options limited still further because anti-EGFR monoclonal antibodies (eg, cetuximab) are ineffective owing to dominant activation of RAS in the MAPK pathway (Douillard et al. 2014). However, preclinical and preliminary clinical data suggest that MAPK pathway inhibition enhances antigen presentation and T-cell cytotoxicity to positively modulate the efficacy of checkpoint inhibitors (Brea et al. 2016; Bendell et al. 2014). The main objective of this open-label multicenter phase 1b/2 study is to evaluate whether the potential positive modulation of NIVO or NIVO plus IPI, when combined with BINI, translates into clinically meaningful overall response in pts with MSS mCRC and RAS mutation. Methods: The study will enroll ~90 previously treated pts (1 or 2 prior regimens), ~42 in phase 1b and ~48 in phase 2. The primary objective of phase 1b will be to determine the recommended phase 2 dose (RP2D) of BINI in combination with NIVO ± IPI. Dose finding in the doublet arm will begin with BINI 45 mg BID + NIVO 480 mg Q4W; the triplet arm will begin with the BINI RP2D from the doublet arm + NIVO 480 mg Q4W + IPI 1 mg/kg Q8W. In phase 2, pts will be randomized 1:1 to doublet or triplet arms, incorporating the BINI RP2Ds found in phase 1b; treatment will continue in 28-day cycles until disease progression, unacceptable toxicity, withdrawal of consent, initiation of subsequent anticancer therapy, loss to follow-up, or death. The primary objective for phase 2 will be to assess response by RECIST version 1.1. The study will also characterize safety and PK. CT.gov Identifier: Clinical trial information: NCT03271047.

Consequence of enhanced LC3-trafficking for a live, attenuated M. tuberculosis vaccine

Development of a new vaccine against tuberculosis is urgently needed. Recent work has demonstrated that two related LC3-associated trafficking pathways, autophagy and LC3-associated phagocytosis (LAP), enhance antigen presentation and might play a role in vaccine efficacy. Mycobacterium tuberculosis inhibits both LC3-trafficking pathways. Moreover, the vaccine strain, BCG, induces even less LC3-trafficking than M. tuberculosis, which may help explain its limited efficacy. To determine whether enhanced LC3-trafficking can improve efficacy of a live, attenuated M. tuberculosis vaccine, we took advantage of our recent finding that the bacterial virulence factor CpsA inhibits LAP. When we deleted cpsA in the mc26206 vaccine strain, it dramatically increased LC3-trafficking. We compared the protective efficacy of the strain lacking cpsA to the parent strain and to BCG in mice challenged with M. tuberculosis. We found that the strain lacking cpsA generated modestly enhanced protection in the spleen, but overall did not outperform BCG.

Signaling by the Epstein–Barr virus LMP1 protein induces potent cytotoxic CD4+ and CD8+ T cell responses

The B-lymphotropic Epstein-Barr virus (EBV), pandemic in humans, is rapidly controlled on initial infection by T cell surveillance; thereafter, the virus establishes a lifelong latent infection in the host. If surveillance fails, fatal lymphoproliferation and lymphomagenesis ensue. The initial T cell response consists of predominantly CD8+ cytotoxic T cells and a smaller expansion of CD4+ cells. A major approach to treating EBV-associated lymphomas is adoptive transfer of autologous or allogeneic T cells that are stimulated/expanded on EBV-transformed B cells. Strikingly, the clinical response correlates with the frequency of CD4 cells in the infused T cells. Although in vitro studies suggested that EBV-specific CD4 cells develop cytotoxicity, they have not been comprehensively characterized and the molecular mechanism underlying their formation remains unknown. Our recent work, using a transgenic approach in mice, has revealed a central role for the EBV signaling molecule LMP1 in immune surveillance and transformation of EBV-infected B cells. The mouse model offers a unique tool for uncovering basic features of EBV immunity. Here, we show that LMP1 expression in B cells induces potent cytotoxic CD4 and CD8 T cell responses, by enhancing antigen presentation and costimulation by CD70, OX40 ligand, and 4-1BB ligand. Our data further suggest that cytotoxic CD4 cells hold superior therapeutic value for LMP1 (EBV)-driven lymphomas. These findings provide insights into EBV immunity, demonstrating that LMP1 signaling alone is sufficient to induce a prominent cytotoxic CD4 response, and suggest strategies for immunotherapy in EBV-related and other cancers.

Low-density lipoprotein receptor–related protein 1 attenuates house dust mite–induced eosinophilic airway inflammation by suppressing dendritic cell–mediated adaptive immune responses

Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

Enhanced antigen presenting and T cell functions during late-phase allergic responses in the lung.

Allergic inflammation is a common feature of asthma and may contribute to both development and perpetuation of disease. The interaction of antigen-presenting cells (APC) with sensitized helper T lymphocytes (TC) producing Th2 cytokines may determine the inflammatory response. Recruitment of APC and TC to the lung during allergic responses has been demonstrated, but functional studies in humans have been limited. This study examined the function of APC and TC accumulating at sites of inflammation after segmental allergen challenge (SAC). Fifteen allergic patients underwent SAC, and cells from bronchoalveolar lavage (BAL) were collected after 24 hours. APC and TC from the blood and BAL were purified based on expression of the monocyte marker, CD14; the plasmacytoid dendritic cell (pDC) marker, BDCA4, identifying neuropilin-1 (NRP1); and the helper T cell marker, CD4. Functional activity was assessed using allergen-induced T cell proliferation. Flow cytometry identified cells expressing CD14 and NRP1. SAC resulted in a 12-fold increase in mononuclear cells having the morphologic appearance of blood monocytes. Most of these cells co-expressed CD14 and NRP1. After saline challenge, BAL mononuclear cells demonstrated little APC function. Following SAC, BAL mononuclear cells showed function equal to pDC from blood and greater than blood monocytes. Purified NRP1+ cells from BAL had even greater function than pDC cells from blood (P = .008). Using consistent sources of APC, enhanced proliferation of TC from lung compared to blood was also demonstrated (P = .002). The marked increase in APC function for allergen-specific TC proliferation during allergic inflammation is largely due to the recruitment of monocytes and dendritic cells. There is also an enhanced response in the lung TC population, consistent with recruitment of allergen-specific T cells. Interactions between recruited APC and TC may occur as an early event promoting allergic airway inflammation.

Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has been treated for decades with cisplatin chemotherapy, and anti-PD-1 immunotherapy has recently been approved for the treatment of this disease. However, preclinical studies of how antitumor immunity in HNSCC is affected by cisplatin alone or in combination with immunotherapies are lacking. Here, we show that sublethal doses of cisplatin may enhance antigen presentation and T-cell killing in vitro, though cisplatin also upregulates tumor cell expression of PD-L1 and may impair T-cell function at higher doses. In a syngeneic mouse model of HNSCC, concurrent use of cisplatin and anti-PD-L1/PD-1 delayed tumor growth and enhanced survival without significantly reducing the number or function of tumor-infiltrating immune cells or increasing cisplatin-induced toxicities. These results suggest that moderate doses of cisplatin may enhance antitumor immunity by mechanisms other than direct tumor cell killing, which may be further enhanced by anti-PD-L1/PD-1 therapy. Cancer Immunol Res; 5(12); 1141-51. ©2017 AACR.

Breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth

Breast cancer immunotherapy is a potent treatment option, with antibody therapies such as trastuzumab increasing 2-year survival rates by 50%. However, active immunotherapy through vaccination has generally been clinically ineffective. One potential means of improving vaccine therapy is by delivering breast cancer antigens to dendritic cells (DCs) for enhanced antigen presentation. To accomplish this in vivo, we pseudotyped lentiviral vector (LV) vaccines with a modified Sindbis Virus glycoprotein so that they could deliver genes encoding the breast cancer antigen alpha-lactalbumin (Lalba) or erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) directly to resident DCs. We hypothesized that utilizing these DC-targeting lentiviral vectors asa breast cancer vaccine could lead to an improved immune response against self-antigens found in breast cancer tumors. Indeed, single injections of the vaccine vectors were able to amplify antigen-specific CD8T cells 4-6-fold over naïve mice, similar to the best published vaccine regimens. Immunization of these mice completely inhibited tumor growth in a foreign antigen environment (LV-ERBB2 in wildtype mice), and it reduced the rate of tumor growth in a self-antigen environment (LV-Lalba in wildtype or LV-ERBB2 in MMTV-huHER2 transgenic). These results show that a single injection with targeted lentiviral vectors can be an effective immunotherapy for breast cancer. Furthermore, they could be combined with other immunotherapeutic regimens to improve outcomes for patients with breast cancer.

PTPN2 Loss Sensitizes Tumor Cells to Immunotherapy

Abstract Ptpn2 loss increases tumor cell IFNγ signaling to enhance antigen presentation and antitumor immunity.

Lactobacillus plantarum Strains Can Enhance Human Mucosal and Systemic Immunity and Prevent Non-steroidal Anti-inflammatory Drug Induced Reduction in T Regulatory Cells.

Orally ingested bacteria interact with intestinal mucosa and may impact immunity. However, insights in mechanisms involved are limited. In this randomized placebo-controlled cross-over trial, healthy human subjects were given Lactobacillus plantarum supplementation (strain TIFN101, CIP104448, or WCFS1) or placebo for 7 days. To determine whether L. plantarum can enhance immune response, we compared the effects of three stains on systemic and gut mucosal immunity, by among others assessing memory responses against tetanus toxoid (TT)-antigen, and mucosal gene transcription, in human volunteers during induction of mild immune stressor in the intestine, by giving a commonly used enteropathic drug, indomethacin [non-steroidal anti-inflammatory drug (NSAID)]. Systemic effects of the interventions were studies in peripheral blood samples. NSAID was found to induce a reduction in serum CD4+/Foxp3 regulatory cells, which was prevented by L. plantarum TIFN101. T-cell polarization experiments showed L. plantarum TIFN101 to enhance responses against TT-antigen, which indicates stimulation of memory responses by this strain. Cell extracts of the specific L. plantarum strains provoked responses after WCFS1 and TIFN101 consumption, indicating stimulation of immune responses against the specific bacteria. Mucosal immunomodulatory effects were studied in duodenal biopsies. In small intestinal mucosa, TIFN101 upregulated genes associated with maintenance of T- and B-cell function and antigen presentation. Furthermore, L. plantarum TIFN101 and WCFS1 downregulated immunological pathways involved in antigen presentation and shared downregulation of snoRNAs, which may suggest cellular destabilization, but may also be an indicator of tissue repair. Full sequencing of the L. plantarum strains revealed possible gene clusters that might be responsible for the differential biological effects of the bacteria on host immunity. In conclusion, the impact of oral consumption L. plantarum on host immunity is strain dependent and involves responses against bacterial cell components. Some strains may enhance specific responses against pathogens by enhancing antigen presentation and leukocyte maintenance in mucosa. In future studies and clinical settings, caution should be taken in selecting beneficial bacteria as closely related strains can have different effects. Our data show that specific bacterial strains can prevent immune stress induced by commonly consumed painkillers such as NSAID and can have enhancing beneficial effects on immunity of consumers by stimulating antigen presentation and memory responses.

Abstract 3649: Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian cancer

Abstract Background and Purpose: Although immunotherapy as an adjuvant to surgery and chemotherapy has been investigated in ovarian cancer (OC) as a means of reducing tumor recurrence and improving survival, there remains a significant unmet need for combinatorial strategies to enhance the antitumor immune response. The purpose of this study was to develop a novel combination immunotherapy for OC, utilizing our novel fusion protein to target and generate a cellular immune response to mesothelin (MSLN) in conjunction with blockade of the PD-1/PD-L1 checkpoint to restore the function of cytotoxic T cells in order to enhance cancer control and prolong survival. Experimental Procedures: Luciferase-expressing ID8 cells were employed to establish an intraperitoneal ovarian tumor model in immunocompetent C57BL/6 mice. The efficacies of the MSLN-targeted immune-activating fusion protein (VIC-008), αPD-L1, and the combination were evaluated. Mice received 4 intraperitoneal (i.p.) treatments of VIC-008 from day 7 post tumor inoculation weekly, and 6 treatments of αPD-L1 i.p. every other day from 4 weeks post inoculation. Tumor growth was monitored by in vivo imaging of luciferase activity. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 7 weeks after tumor cell inoculation. Immune cells from lymph nodes, ascites and tumors were stained with antibodies against multiple immune cell markers and profiled by flow cytometry. Results: VIC-008, αPD-L1 or combination treatment delayed tumor growth. The combination treatment resulted in the greatest prolongation in survival, followed by αPD-L1 treatment and then VIC-008 treatment. Improved survival was associated with increased levels of intratumoral CD3+CD8+ T cells (P<0.0001). The combination treatment also reduced the proportion of CD4+CD25+Foxp3+ Treg cells (P<0.0001) in the lymph nodes. An increased number of CD8+CD27+CD44+ memory T cells (P=0.0134) were observed in ascites in the combination treatment group. CD11b+CD11c+ dendritic cells were enriched in ascites in VIC-008 treatment (P=0.0019) and combination treatment groups (P=0.0010). More CD11c+CD38+ (M1) (P=0.0361) and fewer CD206+CD106+ (M2) (P=0.0285) macrophages were found in the tumors of the combination treatment group. Conclusion: Our results suggest that, through activating dendritic cells and enhancing antigen presentation and cross-presentation, VIC-008 augments antitumor CD8+ T cell responses and facilitates generation of memory T cells when combined with PD-1/PD-L1 blockade, providing long-term antitumor effects. Our findings demonstrate for the first time a mechanistic rationale for combining VIC-008 and αPD-L1 in treatment of OC in mice, positioning this combination therapy as a potential promising new immunotherapeutic approach for OC. Citation Format: Yang Zeng, Binghao Li, Qiuyan Liu, Patrick Reeves, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky, Huabiao Chen. Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian 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 3649. doi:10.1158/1538-7445.AM2017-3649

Abstract 1703: A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming

Abstract Significant clinical and pre-clinical research has shown that PAMPs- Pathogen Associated Molecular Patterns- can trigger an integrated anti-cancer immune response involving both innate and adaptive immunity. Imprime PGG is a soluble yeast β-1,3/1,6 glucan currently in multiple phase 2 clinical studies in combination with the immune checkpoint inhibitor (CPI), pembrolizumab. Preclinical mechanistic research has shown that Imprime PGG can re-polarize immunosuppressive myeloid cells in the tumor microenvironment and activate antigen presenting cells to prime antigen-specific CD8 T cells thereby boosting effector T cell function and expansion. Based on this ability to activate dendritic cells and induce type I interferon, we sought to explore the use of Imprime as an immune activating/antigen-directing scaffold onto which we could attach tumor antigen to drive an antigen-specific T cell based immune response. Imprime PGG has been safely administered intravenously to more than 400 human subjects and thus represents a safe starting point for the development of a cancer vaccine. Imprime’s 1,3/1,6 β-glucan polymeric structure allows for the straightforward attachment of peptides and proteins via three different conjugation routes. To explore this function we covalently linked the chicken ovalbumin (OVA) protein to Imprime to generate a β-glucan-protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8 and OT-II CD4 T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, both OVA-specific CD8 and CD4 T cells underwent vigorous expansion. OT-I CD8 T cells upregulated the transcription factor Tbet, which is central to developing effector functions, and were highly multifunctional in their ability to produce IFN-γ, TNF-α and IL-2. OT-I T cells responded similarly to vaccination with the minimal H-2Kb/OVA257-264 peptide covalently attached to Imprime. By comparison, vaccination with unconjugated OVA (protein or peptide) and Imprime was much less effective in driving T cell expansion and differentiation. The CD8 T cell response required Batf3-dependent cross-presenting DCs whereas the CD4 T cell response did not, and both CD4 and CD8 responses required the C-type lectin receptor Dectin-1. These data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. These data provide evidence that Imprime PGG not only serves as a combination therapy for CPIs, anti-angiogenics, and tumor targeting antibodies but may also be a robust platform for multiple tumor vaccine strategies. Citation Format: Kyle S. Michel, Ross B. Fulton, Steven M. Leonardo, Keith B. Gorden, Jeremy R. Graff, Michael E. Danielson. A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming [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 1703. doi:10.1158/1538-7445.AM2017-1703

Abstract 3652: Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative breast cancer

Abstract Previous studies utilizing NanoString immune profile analysis demonstrated that intratumoral levels of LAG3 (lymphocyte activation gene 3) mRNA increased in response to phosphatidylserine (PS) and PD-1 targeting antibodies in murine triple negative breast cancers (TNBC). This suggests LAG3 acts to attenuate immune system activation during I/O therapies - and that PD-1 and LAG3 function cooperatively in suppressing immune system activation. Here we show that adding PS targeting antibodies can further enhance the effectiveness of antibodies targeting LAG3 and/or LAG3+PD-1. We first examined expression of LAG3 and PD-1 in the murine TNBC model E0771 and found that tumor associated T-cells (CD4+ and CD8+) have expression of both markers. Mice implanted with TNBC tumors were next treated with antibodies targeting PS, PD-1, and LAG3 alone and in combination with each other. Interestingly, the addition of PS targeting antibodies not only increased the effectiveness anti-PD-1 effectiveness as previously observed, but also enhanced anti-LAG3 treatment, showing that PS targeting antibodies are capable of augmenting additional I/O therapeutic regimens. Comparison of anti-PD-1+LAG3 combination vs. single anti-PD-1 or anti-LAG3 treatments showed moderately more anti-tumor activity than single treatments, however the addition of PS targeting antibodies to either checkpoint inhibitor was as equally effective in inhibiting tumor growth as observed in the anti-LAG3+PD-1 treatment. Further comparison of antibody treatments targeting PD-1+LAG3 vs. PS+PD-1+LAG3 demonstrated that the addition of PS targeting antibodies resulted in a significant decrease in tumor growth with complete tumor regression in 80% of the animals (along with the ability to completely reject secondary TNBC challenge) compared to 0% in the anti-PD-1+LAG3 treatment group. Immunoprofiling showed that the addition of PS targeting antibodies to these checkpoint therapies, including the combination of anti-PD-1+LAG3, resulted in a phenotype associated with enhanced immune system activation and immune-surveillance including increased tumor infiltrating lymphocytes (TILs) with upregulation of T-cell associated activation pathways, increased Th1 to Th2 profile, and enhanced antigen presentation processing /presentation mechanisms along with cytokines associated with immune system activation. Overall our data demonstrate that adding PS targeting antibodies to clinically relevant therapies, including PD-1 and LAG3, may significantly enhance their ability to activate and redirect the host immune system into recognition and elimination of tumor cells compared to single and combinational treatments that lack PS targeting antibodies. Citation Format: Michael J. Gray, Jian Gong, Jeff T. Hutchins, Bruce D. Freimark. Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative breast 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 3652. doi:10.1158/1538-7445.AM2017-3652